Mode of action of anti-Candida drugs: Focus on terconazole and other ergosterol biosynthesis inhibitors Hugo Vanden Bossche, ind Ir, and Patrick Marichal, ind Ir Beerse, Belgium A large proportion of the presently available antifungal agents are claimed to derive their activity from interaction with the biosynthesis of ergosterol, the key sterol in most pathogenic fungi. An important target for the allylamines, naftifine and terbinafine, is the squalene epoxidase. Interaction with the epoxidation step results in a decreased availability of ergosterol and an accumulation of squalene. Although the squalene epoxidase is clearly the primary target for this class of antifungals, it still remains an open question whether the fungistatic or fungicidal effects originate from a decrease in ergosterol or squalene accumulation. Indeed, preliminary evidence suggests that squalene does not change the physicochemical properties of membranes. Much more is known about the primary and secondary effects of the azole antifungals, such as miconazole, ketoconazole, terconazole, and itraconazole. Most of the imidazole and triazole derivatives are highly potent and selective inhibitors of the cytochrome P-450-dependent 14u-demethylation of lanosterol (P-450'4DM)' Their potency and selectivity are determined by the nitrogen heterocycle and to a much greater extent by the hydrophobic N-1 substituent. The triazole antifungals, terconazole and itraconazole, combine a high affinity for Candida P-450'4DM with an exceptionally low effect on mammalian cytochrome P-450. (AM J OSSTET GVNECOL 1991 ;165:1193-9.)
Key words: Squalene epoxidase, I4a-demethylase, cytochrome P-450, azoles, allylamines, morpholines
Mode of action studies on antifungals show how these agents interfere with a target in the fungus and highlight the differences between fungi and the mammalian host. Thus these studies may improve our knowledge of the biochemical systems in both parasite and patient. Furthermore, these chemical compounds can be used as tools to investigate the biochemical peculiarities of fungi. Ergosterol is the most common sterol in Candida species and most pathogenic fungi where it plays a major architectural and functional role. Therefore it is not surprising that the ergosterol synthesis pathway has been recognized as a major target of antifungal agents. Indeed, most of the presently available antifungals are claimed to interfere with enzyme systems involved in the synthesis of this 24-alkylated sterol. For example, the allylamines inhibit the squalene epoxidase, the azole antifungals, the cytochrome P-450-dependent I4a-demethylase, and the morpholines interfere with the .1 14 _ reductase and .:ls-7-isomerase (Fig. 1). It is the aim of this overview to discuss the molecular basis of anticandidal action of the squalene epoxidase and 14a-demethylase inhibitors. From the Department of Comparative Biochemistry,janssen Research Foundation. Reprint requests: Hugo Vanden Bossche, ind fr, Department of Comparative Biochemistry,janssen Research Foundation, Tumhoutseweg 30, B2340 Beerse, Belgium. 610132264
Squalene epoxidase inhibitors The allylamines, naftifine and terbinafine, and the thiocarbamates, tolciclate and tolnaftate, induce an accumulation of squalene and a concomitant decrease in ergosterol synthesis in fungal cells. 1 This indicates that both the allylamines and thiocarbamates inhibit the squalene epoxidase (Fig. 1). In dermatophytes there is a clear correlation between the inhibition of this important enzyme of the ergosterol biosynthesis pathway and inhibition of growth. However, although the squalene epoxidases of Candida albicans and Candida parapsilosis are almost equisensitive to terbinafine (95% inhibition at terbinafine concentrations between 0.2 and 0.9 )-lg/ml), the latter species is much more susceptible to growth inhibition. Furthermore, the epoxidase of Candida glabrata is, compared with that of C. parapsilosis, about three times less sensitive to this allylamine, whereas the minimum inhibitory concentration value is 250 times higher. 1 It should be noted that penetration problems do not seem to explain these differences, because activity against squalene epoxidase in intact cells and crude extracts of Candida cells is similar.2 However, poor penetration of the Candida cell membrane accounts for the lack of activity of the thiocarbamates against yeasts. 3 Ryder l speculates that fungal species differ in their sensitivity to a decreased availability of ergosterol and also in their reaction to the intracellular accumulation of squalene. Indeed, the filamentous form of C. albicans 1193
1194
Vanden Bossche and Marchial
October 1991 Am J Obstet Gynecol
HMG·CoA Reductase Acetate
-
Acetyl CoA
-
Mevalonate
HMG-CoA
~ I
I
o
AIlYlamine.~,;P' I ( ......
Thiocarbamat..
.
I
I ~
Squalene epoxidase
I
2.3-oxidosquaJene
2,3:22.23-dioxidosqualene
Squalene
¢e' """'~'""'" Squalene cyclase
H~
~ '
'.H
H
•
HO
.. H , 24-methylenedihydrolanosterol
f
-
HO
'~
14
""'"
:
H
'~
2'
: I
""'"
AzoleJ
.. H I HO " H '14,,·Oemethylase·
Lanosterol
(P450)
.1 14 . Reductss.
-
t
Morpholines
4a'DBmBthJIBSB (P450)
Azole.
,
H~ .H
~ '
d
HO
HO
...
,
Hr .H
2'~'
. H .:.H
Q1S'~I
Morphohn ••
".Redu~rBSe
H
""'"
HO
HO
'cA
... H
4
' ds? I
I
I
cA~ l
HoctT : H
Ergosterol
... H
HoctT : H
"'H~ ~' ".H~
Morpholin ••
~'_L17.ls~se HO
.-----
H
Episterol
Multistep reac1ion
HO
I
H
Fecosterol
'"
HO
Zymosterol
H
Inhibition
Fig. 1. Reaction sequences involved in the synthesis of ergosterol and target sites for all ylamines, thiocarbamates, azole antifungals, and morpholines.
is more susceptible than the yeast form, whereas ergosterol biosynthesis is almost equally affected in both forms. Filamentous fungi seem particularly susceptible to the allylamines and require only partial ergosterol biosynthesis inhibition for full inhibition of growth. Does the sensitivity to the accumulating squalene determine the susceptibility? Candida species can grow semianaerobically. Under this condition low amounts of ergosterol are formed and high levels of squalene are found! Thus Candida. seems to be able to tolerate high amounts of squalene. It has been speculated that filamentous fungi are more susceptible to squalene accumulation. However, of interest is the high radioactivity incorporated from carbon 14 acetate into squa-
lene by Trichophyton mentagrophytes grown under control conditions! Almost 24% of the total radioactivity measured in the lipid extracts is found in the squalene fraction; this is only two times less than the radioactivity incorporated into ergosterol.' The presence of squalene did not prevent growth" This is not surprising because microgram amounts of squalene have been found in Phytophothora cactorum, a fungus unable to epoxidise and cyclisize squalene to lanosterol,5.6 and Margalith 7 hypothesized that in Phytophothora "squalene itself may be assuming the architectural role (bulk function?) of sterols, allowing vegetative growth." Differential scanning calorimetry of multilamellar vesicles of dipalmitoylphosphatidylcholine containing increasing
Anti-Candida drugs: Focus on ergosterol
Volume 165 !'lumber 4, Part 2
concentrations of squalene (10 to 35 mol %) has shown that squalene, even at 35 mol %, induced no significant change « 1 C) of the dipalmitoylphosphatidylcholine mid transition temperature or the enthalpy of melting (unpublished results). This suggests that squalene does not change the physicochemical properties of the membranes and thus differs from its cyclic products formed by the squalene cyclase, such as lanosterol (Fig. I). Indeed, the addition of lanosterol (5 to 15 mol %) to dipalmitoylphosphatidylcholine multilamellar vesicles lowered the transition temperature slightly but had a major effect on the enthalpy of melting," suggesting that lanosterol interacts with the phospholipids present. Therefore it is still an open question whether growth inhibition and especially fungicidal activity are consequences of the decreased availability of ergosterol, squalene accumulation, or both. Thus although the squalene epoxidase is clearly a target for the allylamines, more studies are needed to clarify the molecular basis for their fungistatic and fungicidal activity. 0
14a-Demethylase inhibitors
Since the synthesis of miconazole and clotrimazole in the 1960s, a huge number of azole derivatives have become available. Some are used topically, whereas others are used both topically and orally. All belong to the class of 14a-demethylase inhibitors."' '0 Their antifungal activity originates from binding to a cytochrome P-450 (P-450"nM) involved in the 14a-demethylation of lanosterol (in Saccharomyces cerevi~iae, C. glabrata, and mammalian cells) or 24-methylenedihydrolanosterol (in filamentous fungi, the yeast form of Hi~toplasma capsulatum, Cryptococcus neofonnam, and a number of C. albicam isolates) (Fig. I). It should be noted that contrary to C. albicam, C. glabrata uses lanosterol preferentially as substrate. Before a discussion of the interaction of the azole antifungals with this key enzyme of sterol biosynthesis, we should look at cytochromes P-450, which are present in just about every phyla in which they have been sought. They are membrane bound (mitochondrial inner membrane and endoplasmic reticulum) in eukaryotes, whereas the prokaryote forms are soluble proteins. Cytochrome P-450s catalyze the synthesis or metabolism of a long list of key compounds. Examples are sterols (e.g., ergosterol and cholesterol), steroids (e.g., androgens and estrogens), bile acids, vitamin A, thromboxane A2, prostacyclin, and leukotrienes." They also play an important role in the activation of vitamin 0 and the metabolism of xenobiotics. Most of the cytochrome P-450s are classified as monooxygenases. This means that they catalyze the following reaction: NADPH 2 + O 2 + RH NADP+ + H 20 + ROH. In this reaction, RH represents a substrate (e.g., lanosterol) to be oxidized by the P-450 enzyme. The reaction
=>
1195
requires molecular oxygen of which one oxygen atom is inserted into the substrate, whereas the other oxygen atom is reduced to water. NADPH 2 provides the electrons needed for the activation of this process. The active site of P-450 contains a ferric prosthetic heme group, the substrate binds to the protein moiety of the P-4S0, and the heme iron is reduced. Molecular oxygen is bound, reduced, and activated at this site (Fig. 2, A and B). The specific enzymatic function of the cytochrome P-450s, that is, to activate oxygen for insertion into a substrate, originates from the electronic structure of the heme iron linked to the thiol of a cysteyl residue of the P-450 protein moiety (Fig. 2, A). The typical absorption maximum at 450 nm of the reduced carbon monoxide-P-450 complex also originates from the specific interaction of the prosthetic group with the protein. The name of this hemoprotein originates from this property. Indeed, the name cytochrome P-450 describes a carbon-monoxide-binding pigment, which absorbs at about 450 nm. As shown in Fig. 2, B, azole antifungals bind to the heme iron and compete in this way with oxygen binding and activation. This results in inhibition of the P-450catalyzed reaction. Because carbon monoxide also competes with oxygen for the same binding place, we can measure the interaction of an azole antifungal by adding it to a cytochrome P-450-containing microsomal or mitochondrial fraction, reducing the heme iron (with dithionite), and bubbling the suspension with carbon monoxide. Binding of the inhibitor to the heme iron will reduce absorption at 450 nm, and this decrease is a measure for the amount of inhibitor bound. For example, 50% inhibition of carbon monoxide binding to microsomal P-450 from C. albicam is obtained with nanomolar concentrations of the imidazole derivatives, miconazole and ketoconazole, and the triazoles, terconazole and itraconazole (Table I). Another triazole derivative, fluconazole. is a much less potent inhibitor of carbon monoxide binding, indicating that it has a lower affinity for the Candida P-450. This already indicates that the potency of an azole derivative is determined not only by its binding to the heme iron. It has been shown previously that the activity is determined, at least partly, by the hydrophobicity of the N-I substituent (Fig. 2, B).'2 For example, itraconazole and its imidazole analog form equistable complexes with C. albicam P-450; the less hydrophobic ketoconazole forms a less stable P-450 complex, and the stability of the complex is not increased by replacing the imidazole by a triazole (R42164).'2 Comparing the amino-acid sequences of mammalian P-450(s) (e.g., those involved in steroid synthesis and metabolism) with that of C. albicam P-4S0'.'OM shows less than 25% identical amino acids.'" Therefore it should
1196 Vanden Bossche and Marchial
October 1991 Am J Obstet Gyneco1
Fig. 2. A, Sketch of part of cytochrome P-450 14DM • Iron protoporphyrin and substrate (lanosterol) bound to a hydrophobic domain of the polypeptide chain are shown. The iron (Fe'+) is linked to the thiol of a cysteyl residue (C) that is part of a conserved region of the polypeptide in C. albicans P-450 14DM H, Histidine; R, arginine; I, isoleucine; G, glycine. Molecular oxygen is attached to the ferrous heme iron. B, Sketch of azole (terconazole)-P-450 complex. Triazole ring is linked to the sixth position of the ferric (Fe 3 +) heme iron by way of N-3. N-l substituent is bound to a hydrophobic binding site in P-450.
be possible to synthesize selective anti-Candida compounds. The selectivity of the azole antifungals may be determined by both the azole and the N-l substituent of the molecule. Terconazole and itraconazole (triazole derivatives) are, as shown in Fig. 3, more selective affectors of the yeast P-450s than the imidazole antifungals miconazole, clotrimazole, and bifonazole. However, major differences in selectivity are found among
the topically active imidazole antifungals. 1o• 13 For example, clotrimazole and miconazole interact preferentially with the Candida P-450, whereas bifonazole has highest affinity for P-450(s) of the testicular microsomes from piglets (Fig. 3). Replacing the imidazole ring of ketoconazole with a triazole ring (R42l64) slightly decreases the interaction with the microsomal P-450(s) from piglet testes. 13 Fifty percent inhibition is
Anti-Candida drugs: Focus on ergosterol
Volume 165 Number 4, Part 2
*
*
10
*
1197
*
1
tn
C1> ::l
ca
0 IZI tzJ 0
> I
o
LO
U
.1
~
El
• .01
s. cerevisiae c. albicans C. glabrata Ad. Mito. Testes Liver PB liver
• = >10 11 M
CTZ
BFZ
MCZ
TCZ
ITZ
Fig. 3. Effects of clotrimazole (CTl), bifonazole (RFl), miconazo le (MCl), terconazole (TCl), and itraconazole (ITl) on microsomal P-450s from Saccharomyces cerevisiae (S. cerevisiae), C. albicans, C. glabrata, piglet testes, and liver from untreated and phenobarbital (PR) pretreated rabbits, and on mitochondrial P-450 from bovine adrenal glands (ad. mito). ICso values = drug concentrations needed to inhibit carbon monoxide binding the heme iron by 50%. P·450 content = 0. 1 nmoJ . ml - '. Methods used are desCTibed in ref. 20.
Table I. Effects of azole antifungals on carbon monoxide-binding to microsomal P-450 and on ergosterol synthesis IC w values (nmQlI L)
P-450* Antifungals
C. albicans
Miconazole Ketoconazole Terconazole Itraconazole Fluconazole
93.5 31.1 41.0 30.1 248.0
I
Ergosterol synthesist
C. glabrata
C. albicans
44.6 30.4 76.7 35.6 260.0
II 7 60.3 26.8 80.3 6530.0
I
C. glabrata 56.7 52.8 65.7 40.5 1790.0
IC .. values = concentrations needed to achieve 50% decrease in tlA (448 nm - 490 nm). *Membranes used were isolated from C. albicans (ATCC 28516, microsomes) and C. glabrata (B 16205, microsomes). t Cells were first grown for 16 hours (8 hours in an orbital shaker) at 30° C in a poly peptone : yeast extract: glucose (10: 10 :40 gmLlL) medium and then washed and resuspended in a 0.1 nmollL potassium phosphate buffer containing 56 nmollL glucose (pH 6.5). ['4C)-Acetate, azole antifungals, and /or dimethylsulfoxide were added, and the cell suspensions were incubated for 2 hours at 30° C in an orbital shaker (300 rpm). At the end of the incubation period, sterols were extracted and separated by thinlayer chromatography. I Cso values: Concentrations needed to obtain 50% inhibition of [14C)-incorporation into ergosterol.
achieved at 0.39 fJ-moIlL (ketoconazole) and 0.49 fJ-mol/L (R42164). Replacing the triazole of itraconazole with an imidazole ring slightly increased the effects on adrenal mitochondrial P-450 (from 0% to 25% inhibition at 1 fJ-moIlL) but did not enhance the effects on testicular microsomal P-450.13 Furthermore, comparing the effects of ketoconazole with those of nor-
ketoconazole (deacylated ketoconazole) indicates that minor structural changes in the nonligand part (the N- l substituent) affects the interaction with P-450. Norketoconazole has two- and three-times lower affinity for the microsomal P-450 from piglet testis and C. albicans, respectively. '3 These results suggest that the nonligand hydrophobic part of the azoles has a greater impact on
October 1991 Am J Obstet Gynecol
1198 Vanden Bossche and Marchial
% of total of radioactivit~
Terconazole
Sterol (0)
(1 J.lM)
76.9
0.0
0.0
59.4
14a-Methylfecosterol
0.0
6.2
Obtusifoliol
0.0
8.2
Lanosterol
0.0
5.9
24-Methylenedihydrolanosterol
0.0
6.2
Ergosterol HO
14a-Methyl-ergosta~8,24(28)-dien-
3f3,6a-diol
Fig. 4. Effects of terconazole on ergosterol synthesis in C. albicans grown for 16 hours in casein hydrolysate, yeast extract, glucose medium supplemented with 14C-acetate and terconazole and/or dimethylsulfoxide. Homogenization of the cells, saponification, extraction, and separation of the sterols by high-performance liquid chromatography was described previously.14 [14C]cAcetate and drug (l fJ.moIlL) and/or solvent were added at inoculation. 0 = the control. Under the conditions used, an IC so value of 45.9 nmollL was found.
the selective interaction with P-450 enzymes than does the azole moiety and also prove that it is possible to synthesize highly potent and selective P-450 inhibitors. The majorP-450 present in C. albicans microsomes is involved in the 14a-demethylation of lanosterol or 24-methylenedihydrolanosterol, that is, P-450 14DM . Thus the effects of azole antifungals are suggestive of an interaction with the 14a-demethylase and thus with ergosterol synthesis. Inhibition of ergosterol biosynthesis has been shown with miconazole, terconazole, ketoconazole, itraconazole, and/ or saperconazole in C. albicans, C. glabrata, Candida lusitaniae, Pityrosporum ovale, T. mentagrophytes, Paracoccidioides brasiliensis, Histoplasma capsulatum, and/or Aspergillus fumigatus.
Key words: Squalene epoxidase, I4a-demethylase, cytochrome P-450, azoles, allylamines, morpholines
Mode of action studies on antifungals show how these agents interfere with a target in the fungus and highlight the differences between fungi and the mammalian host. Thus these studies may improve our knowledge of the biochemical systems in both parasite and patient. Furthermore, these chemical compounds can be used as tools to investigate the biochemical peculiarities of fungi. Ergosterol is the most common sterol in Candida species and most pathogenic fungi where it plays a major architectural and functional role. Therefore it is not surprising that the ergosterol synthesis pathway has been recognized as a major target of antifungal agents. Indeed, most of the presently available antifungals are claimed to interfere with enzyme systems involved in the synthesis of this 24-alkylated sterol. For example, the allylamines inhibit the squalene epoxidase, the azole antifungals, the cytochrome P-450-dependent I4a-demethylase, and the morpholines interfere with the .1 14 _ reductase and .:ls-7-isomerase (Fig. 1). It is the aim of this overview to discuss the molecular basis of anticandidal action of the squalene epoxidase and 14a-demethylase inhibitors. From the Department of Comparative Biochemistry,janssen Research Foundation. Reprint requests: Hugo Vanden Bossche, ind fr, Department of Comparative Biochemistry,janssen Research Foundation, Tumhoutseweg 30, B2340 Beerse, Belgium. 610132264
Squalene epoxidase inhibitors The allylamines, naftifine and terbinafine, and the thiocarbamates, tolciclate and tolnaftate, induce an accumulation of squalene and a concomitant decrease in ergosterol synthesis in fungal cells. 1 This indicates that both the allylamines and thiocarbamates inhibit the squalene epoxidase (Fig. 1). In dermatophytes there is a clear correlation between the inhibition of this important enzyme of the ergosterol biosynthesis pathway and inhibition of growth. However, although the squalene epoxidases of Candida albicans and Candida parapsilosis are almost equisensitive to terbinafine (95% inhibition at terbinafine concentrations between 0.2 and 0.9 )-lg/ml), the latter species is much more susceptible to growth inhibition. Furthermore, the epoxidase of Candida glabrata is, compared with that of C. parapsilosis, about three times less sensitive to this allylamine, whereas the minimum inhibitory concentration value is 250 times higher. 1 It should be noted that penetration problems do not seem to explain these differences, because activity against squalene epoxidase in intact cells and crude extracts of Candida cells is similar.2 However, poor penetration of the Candida cell membrane accounts for the lack of activity of the thiocarbamates against yeasts. 3 Ryder l speculates that fungal species differ in their sensitivity to a decreased availability of ergosterol and also in their reaction to the intracellular accumulation of squalene. Indeed, the filamentous form of C. albicans 1193
1194
Vanden Bossche and Marchial
October 1991 Am J Obstet Gynecol
HMG·CoA Reductase Acetate
-
Acetyl CoA
-
Mevalonate
HMG-CoA
~ I
I
o
AIlYlamine.~,;P' I ( ......
Thiocarbamat..
.
I
I ~
Squalene epoxidase
I
2.3-oxidosquaJene
2,3:22.23-dioxidosqualene
Squalene
¢e' """'~'""'" Squalene cyclase
H~
~ '
'.H
H
•
HO
.. H , 24-methylenedihydrolanosterol
f
-
HO
'~
14
""'"
:
H
'~
2'
: I
""'"
AzoleJ
.. H I HO " H '14,,·Oemethylase·
Lanosterol
(P450)
.1 14 . Reductss.
-
t
Morpholines
4a'DBmBthJIBSB (P450)
Azole.
,
H~ .H
~ '
d
HO
HO
...
,
Hr .H
2'~'
. H .:.H
Q1S'~I
Morphohn ••
".Redu~rBSe
H
""'"
HO
HO
'cA
... H
4
' ds? I
I
I
cA~ l
HoctT : H
Ergosterol
... H
HoctT : H
"'H~ ~' ".H~
Morpholin ••
~'_L17.ls~se HO
.-----
H
Episterol
Multistep reac1ion
HO
I
H
Fecosterol
'"
HO
Zymosterol
H
Inhibition
Fig. 1. Reaction sequences involved in the synthesis of ergosterol and target sites for all ylamines, thiocarbamates, azole antifungals, and morpholines.
is more susceptible than the yeast form, whereas ergosterol biosynthesis is almost equally affected in both forms. Filamentous fungi seem particularly susceptible to the allylamines and require only partial ergosterol biosynthesis inhibition for full inhibition of growth. Does the sensitivity to the accumulating squalene determine the susceptibility? Candida species can grow semianaerobically. Under this condition low amounts of ergosterol are formed and high levels of squalene are found! Thus Candida. seems to be able to tolerate high amounts of squalene. It has been speculated that filamentous fungi are more susceptible to squalene accumulation. However, of interest is the high radioactivity incorporated from carbon 14 acetate into squa-
lene by Trichophyton mentagrophytes grown under control conditions! Almost 24% of the total radioactivity measured in the lipid extracts is found in the squalene fraction; this is only two times less than the radioactivity incorporated into ergosterol.' The presence of squalene did not prevent growth" This is not surprising because microgram amounts of squalene have been found in Phytophothora cactorum, a fungus unable to epoxidise and cyclisize squalene to lanosterol,5.6 and Margalith 7 hypothesized that in Phytophothora "squalene itself may be assuming the architectural role (bulk function?) of sterols, allowing vegetative growth." Differential scanning calorimetry of multilamellar vesicles of dipalmitoylphosphatidylcholine containing increasing
Anti-Candida drugs: Focus on ergosterol
Volume 165 !'lumber 4, Part 2
concentrations of squalene (10 to 35 mol %) has shown that squalene, even at 35 mol %, induced no significant change « 1 C) of the dipalmitoylphosphatidylcholine mid transition temperature or the enthalpy of melting (unpublished results). This suggests that squalene does not change the physicochemical properties of the membranes and thus differs from its cyclic products formed by the squalene cyclase, such as lanosterol (Fig. I). Indeed, the addition of lanosterol (5 to 15 mol %) to dipalmitoylphosphatidylcholine multilamellar vesicles lowered the transition temperature slightly but had a major effect on the enthalpy of melting," suggesting that lanosterol interacts with the phospholipids present. Therefore it is still an open question whether growth inhibition and especially fungicidal activity are consequences of the decreased availability of ergosterol, squalene accumulation, or both. Thus although the squalene epoxidase is clearly a target for the allylamines, more studies are needed to clarify the molecular basis for their fungistatic and fungicidal activity. 0
14a-Demethylase inhibitors
Since the synthesis of miconazole and clotrimazole in the 1960s, a huge number of azole derivatives have become available. Some are used topically, whereas others are used both topically and orally. All belong to the class of 14a-demethylase inhibitors."' '0 Their antifungal activity originates from binding to a cytochrome P-450 (P-450"nM) involved in the 14a-demethylation of lanosterol (in Saccharomyces cerevi~iae, C. glabrata, and mammalian cells) or 24-methylenedihydrolanosterol (in filamentous fungi, the yeast form of Hi~toplasma capsulatum, Cryptococcus neofonnam, and a number of C. albicam isolates) (Fig. I). It should be noted that contrary to C. albicam, C. glabrata uses lanosterol preferentially as substrate. Before a discussion of the interaction of the azole antifungals with this key enzyme of sterol biosynthesis, we should look at cytochromes P-450, which are present in just about every phyla in which they have been sought. They are membrane bound (mitochondrial inner membrane and endoplasmic reticulum) in eukaryotes, whereas the prokaryote forms are soluble proteins. Cytochrome P-450s catalyze the synthesis or metabolism of a long list of key compounds. Examples are sterols (e.g., ergosterol and cholesterol), steroids (e.g., androgens and estrogens), bile acids, vitamin A, thromboxane A2, prostacyclin, and leukotrienes." They also play an important role in the activation of vitamin 0 and the metabolism of xenobiotics. Most of the cytochrome P-450s are classified as monooxygenases. This means that they catalyze the following reaction: NADPH 2 + O 2 + RH NADP+ + H 20 + ROH. In this reaction, RH represents a substrate (e.g., lanosterol) to be oxidized by the P-450 enzyme. The reaction
=>
1195
requires molecular oxygen of which one oxygen atom is inserted into the substrate, whereas the other oxygen atom is reduced to water. NADPH 2 provides the electrons needed for the activation of this process. The active site of P-450 contains a ferric prosthetic heme group, the substrate binds to the protein moiety of the P-4S0, and the heme iron is reduced. Molecular oxygen is bound, reduced, and activated at this site (Fig. 2, A and B). The specific enzymatic function of the cytochrome P-450s, that is, to activate oxygen for insertion into a substrate, originates from the electronic structure of the heme iron linked to the thiol of a cysteyl residue of the P-450 protein moiety (Fig. 2, A). The typical absorption maximum at 450 nm of the reduced carbon monoxide-P-450 complex also originates from the specific interaction of the prosthetic group with the protein. The name of this hemoprotein originates from this property. Indeed, the name cytochrome P-450 describes a carbon-monoxide-binding pigment, which absorbs at about 450 nm. As shown in Fig. 2, B, azole antifungals bind to the heme iron and compete in this way with oxygen binding and activation. This results in inhibition of the P-450catalyzed reaction. Because carbon monoxide also competes with oxygen for the same binding place, we can measure the interaction of an azole antifungal by adding it to a cytochrome P-450-containing microsomal or mitochondrial fraction, reducing the heme iron (with dithionite), and bubbling the suspension with carbon monoxide. Binding of the inhibitor to the heme iron will reduce absorption at 450 nm, and this decrease is a measure for the amount of inhibitor bound. For example, 50% inhibition of carbon monoxide binding to microsomal P-450 from C. albicam is obtained with nanomolar concentrations of the imidazole derivatives, miconazole and ketoconazole, and the triazoles, terconazole and itraconazole (Table I). Another triazole derivative, fluconazole. is a much less potent inhibitor of carbon monoxide binding, indicating that it has a lower affinity for the Candida P-450. This already indicates that the potency of an azole derivative is determined not only by its binding to the heme iron. It has been shown previously that the activity is determined, at least partly, by the hydrophobicity of the N-I substituent (Fig. 2, B).'2 For example, itraconazole and its imidazole analog form equistable complexes with C. albicam P-450; the less hydrophobic ketoconazole forms a less stable P-450 complex, and the stability of the complex is not increased by replacing the imidazole by a triazole (R42164).'2 Comparing the amino-acid sequences of mammalian P-450(s) (e.g., those involved in steroid synthesis and metabolism) with that of C. albicam P-4S0'.'OM shows less than 25% identical amino acids.'" Therefore it should
1196 Vanden Bossche and Marchial
October 1991 Am J Obstet Gyneco1
Fig. 2. A, Sketch of part of cytochrome P-450 14DM • Iron protoporphyrin and substrate (lanosterol) bound to a hydrophobic domain of the polypeptide chain are shown. The iron (Fe'+) is linked to the thiol of a cysteyl residue (C) that is part of a conserved region of the polypeptide in C. albicans P-450 14DM H, Histidine; R, arginine; I, isoleucine; G, glycine. Molecular oxygen is attached to the ferrous heme iron. B, Sketch of azole (terconazole)-P-450 complex. Triazole ring is linked to the sixth position of the ferric (Fe 3 +) heme iron by way of N-3. N-l substituent is bound to a hydrophobic binding site in P-450.
be possible to synthesize selective anti-Candida compounds. The selectivity of the azole antifungals may be determined by both the azole and the N-l substituent of the molecule. Terconazole and itraconazole (triazole derivatives) are, as shown in Fig. 3, more selective affectors of the yeast P-450s than the imidazole antifungals miconazole, clotrimazole, and bifonazole. However, major differences in selectivity are found among
the topically active imidazole antifungals. 1o• 13 For example, clotrimazole and miconazole interact preferentially with the Candida P-450, whereas bifonazole has highest affinity for P-450(s) of the testicular microsomes from piglets (Fig. 3). Replacing the imidazole ring of ketoconazole with a triazole ring (R42l64) slightly decreases the interaction with the microsomal P-450(s) from piglet testes. 13 Fifty percent inhibition is
Anti-Candida drugs: Focus on ergosterol
Volume 165 Number 4, Part 2
*
*
10
*
1197
*
1
tn
C1> ::l
ca
0 IZI tzJ 0
> I
o
LO
U
.1
~
El
• .01
s. cerevisiae c. albicans C. glabrata Ad. Mito. Testes Liver PB liver
• = >10 11 M
CTZ
BFZ
MCZ
TCZ
ITZ
Fig. 3. Effects of clotrimazole (CTl), bifonazole (RFl), miconazo le (MCl), terconazole (TCl), and itraconazole (ITl) on microsomal P-450s from Saccharomyces cerevisiae (S. cerevisiae), C. albicans, C. glabrata, piglet testes, and liver from untreated and phenobarbital (PR) pretreated rabbits, and on mitochondrial P-450 from bovine adrenal glands (ad. mito). ICso values = drug concentrations needed to inhibit carbon monoxide binding the heme iron by 50%. P·450 content = 0. 1 nmoJ . ml - '. Methods used are desCTibed in ref. 20.
Table I. Effects of azole antifungals on carbon monoxide-binding to microsomal P-450 and on ergosterol synthesis IC w values (nmQlI L)
P-450* Antifungals
C. albicans
Miconazole Ketoconazole Terconazole Itraconazole Fluconazole
93.5 31.1 41.0 30.1 248.0
I
Ergosterol synthesist
C. glabrata
C. albicans
44.6 30.4 76.7 35.6 260.0
II 7 60.3 26.8 80.3 6530.0
I
C. glabrata 56.7 52.8 65.7 40.5 1790.0
IC .. values = concentrations needed to achieve 50% decrease in tlA (448 nm - 490 nm). *Membranes used were isolated from C. albicans (ATCC 28516, microsomes) and C. glabrata (B 16205, microsomes). t Cells were first grown for 16 hours (8 hours in an orbital shaker) at 30° C in a poly peptone : yeast extract: glucose (10: 10 :40 gmLlL) medium and then washed and resuspended in a 0.1 nmollL potassium phosphate buffer containing 56 nmollL glucose (pH 6.5). ['4C)-Acetate, azole antifungals, and /or dimethylsulfoxide were added, and the cell suspensions were incubated for 2 hours at 30° C in an orbital shaker (300 rpm). At the end of the incubation period, sterols were extracted and separated by thinlayer chromatography. I Cso values: Concentrations needed to obtain 50% inhibition of [14C)-incorporation into ergosterol.
achieved at 0.39 fJ-moIlL (ketoconazole) and 0.49 fJ-mol/L (R42164). Replacing the triazole of itraconazole with an imidazole ring slightly increased the effects on adrenal mitochondrial P-450 (from 0% to 25% inhibition at 1 fJ-moIlL) but did not enhance the effects on testicular microsomal P-450.13 Furthermore, comparing the effects of ketoconazole with those of nor-
ketoconazole (deacylated ketoconazole) indicates that minor structural changes in the nonligand part (the N- l substituent) affects the interaction with P-450. Norketoconazole has two- and three-times lower affinity for the microsomal P-450 from piglet testis and C. albicans, respectively. '3 These results suggest that the nonligand hydrophobic part of the azoles has a greater impact on
October 1991 Am J Obstet Gynecol
1198 Vanden Bossche and Marchial
% of total of radioactivit~
Terconazole
Sterol (0)
(1 J.lM)
76.9
0.0
0.0
59.4
14a-Methylfecosterol
0.0
6.2
Obtusifoliol
0.0
8.2
Lanosterol
0.0
5.9
24-Methylenedihydrolanosterol
0.0
6.2
Ergosterol HO
14a-Methyl-ergosta~8,24(28)-dien-
3f3,6a-diol
Fig. 4. Effects of terconazole on ergosterol synthesis in C. albicans grown for 16 hours in casein hydrolysate, yeast extract, glucose medium supplemented with 14C-acetate and terconazole and/or dimethylsulfoxide. Homogenization of the cells, saponification, extraction, and separation of the sterols by high-performance liquid chromatography was described previously.14 [14C]cAcetate and drug (l fJ.moIlL) and/or solvent were added at inoculation. 0 = the control. Under the conditions used, an IC so value of 45.9 nmollL was found.
the selective interaction with P-450 enzymes than does the azole moiety and also prove that it is possible to synthesize highly potent and selective P-450 inhibitors. The majorP-450 present in C. albicans microsomes is involved in the 14a-demethylation of lanosterol or 24-methylenedihydrolanosterol, that is, P-450 14DM . Thus the effects of azole antifungals are suggestive of an interaction with the 14a-demethylase and thus with ergosterol synthesis. Inhibition of ergosterol biosynthesis has been shown with miconazole, terconazole, ketoconazole, itraconazole, and/ or saperconazole in C. albicans, C. glabrata, Candida lusitaniae, Pityrosporum ovale, T. mentagrophytes, Paracoccidioides brasiliensis, Histoplasma capsulatum, and/or Aspergillus fumigatus.
