Direct Interaction of Antifungal Azole-Derivatives with Calmodulin: a Possible Mechanism for Their Therapeutic Activity

Direct Interaction of Antifungal Azole-Derivatives with Calmodulin: A Possible Mechanism for Their Therapeutic Activity

Lutz Hegemann, Susan M. Toso, Khosrow I. Lahijani, Guy F. Webster, and Jouni Ditto Departments of Dermatology, and Biochemistry and Molecular Biology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania, U.S.A.

Azole derivatives, such as ketoconazole and bifonazole, are competitively inhibited calmodulin activity with 50% inhibi­ well-established antifungal drugs. Recently, these com­ tory concentration values in the low micromolar range. In pounds have been reported to have therapeutic efficacy also contrast, antifungal drugs belonging to other chemical in inflammatory skin disorders. There is increasing evidence classes did not display inhibitory activity. Thus, this study that calmodulin is involved in fungal infections as well as in provides evidence that direct interaction with calmodulin inflammatory skin diseases. Therefore, we investigated the might contribute to the therapeutic activity of azole deriva­ effects of various antifungal drugs on calmodulin activity, tives, particularly to their efficacy in the treatment of inflam­ using calmodulin-dependent phosphodiesterase as an indica­ matory skin disorders. ] I,1vest Dermatol1 00:343 -346, 1993 tor for the calmodulin activity. All azole derivatives tested

zole derivatives, such as bifonazole and ketoconazole, tives and six non-azole antifungals, to inhibit CaM activity, using have long been used as potent antifungal drugs (for CaM-dependent cyclic 3': 5'-nucleotide phosphodiesterase (PDE) reviews, see [1,2]). In recent case reports and clinical as an indicator enzyme. trials, bifonazole and ketoconazole have also been re­ MATERIALS AND METHODS ported to be of therapeutic value in inflammatory skin Adiseases, such as seborrheic dermatitis [3-10]. Furthermore, bifona­ CaM activity was measured based on its activation of cyclic 3' : 5'­ zole has been shown to exert direct anti-inflammatory activity in nucleotide phosphodiesterase (CaM-dependent PDE) from beef histamine-induced erythema [11]. The mechanism of action sug­ heart (Boehringer Mannheim, Indianapolis, IN), using [3H]adeno­ gested for the antifungal activity of azole derivatives is the inhibi­ sine 3' : 5'-cyclic monophosphate as substrate (NEN Du Pont, Wil­ tion of ergosterol biosynthesis, an essential step in the membrane mington, DE), as previously described in detail [21]. CaM-indepen­ formation of fungal cells, resulting in the fungistatic activity of dent PDE from the same donor tissue (Boehringer Mannheim, azole derivatives [12]. However, this mechanism cannot explain the Indianapolis, IN) was used as a control. In the case of CaM-depen­ direct anti-inflammatory properties of the azole derivatives, particu­ de.nt PDE, th~ assay was performed in the presence of 10 ng CaM larly of bifonazole, which have been observed in clinical trials (Sigma Chemical Co., St. LoUls, MO) in 500,u1 incubation volume, [10,11,13] and animal studies [14]. unless otherwise indicated. This CaM concentration yielded about Calmodulin (CaM), a calcium-binding protein that shows a 80% of the maximum enzyme activity. After an incubation period highly conserved structure in different tissues and in a large variety of 20 min at 37 0 C, the reaction was terminated by addition of zinc of species, is involved in the regulation of various cellular responses, sulf~te.' and the.reactio.n product, adenosine 5'-monophosphate, was including prostaglandin synthesis and histamine release from mast preCipitated With banum hydroxide according to the method of cells (for review, see [15]). Furthermore, increased levels of CaM Pbch [22]. ~ftercel:tri~ugation, the remaining substrate was quanti­ have been shown to be associated with dermatologic diseases char­ fied by hqUld scmttllatlon counting. acterized by inflammation of the skin, such as psoriasis [16,17]. In The antifungal drugs tested were dissolved in dimethyl sulfoxide recent studies, evidence has been obtained that CaM might be in­ (DMSO), kept under light protection, and diluted to a final DMSO volved in the regulation of yeast-mycelial transition of Candida concentration of 0.1 % in the assay. This concentration of DMSO albicans [18,19]. This morphogenesis is thought to be a critical step did .not affect .the enzyme activity. Each drug was tested in concen­ in the pathogenesis of fungal infections [20]. Accordingly, we have trations rangmg from O.l,uM to 1 mM. All incubations were investigated the ability of various antifungal drugs, five azole deriva- performed in triplicate and the drugs were tested in at least three independent experiments. All drugs, with the exception of keto­ conazole, were obtained from Sigma Chemical Co. (St. Louis, MO). Manuscript received July 22, 1992; accepted for publication December Ketoconazole was a generous gift of The Janssen Research Founda­ 8, 1992. tion (Beerse, Belgium). Reprint requests to: Dr. Lutz Hegemann, Department of Dermatology, University of Vienna Medical School, Wiihringer Giirtel 18-20, 1090 RESULTS Vienna, Austria. Abbrevia tions: Various antifungal drugs with different chemical structures were CaM: calmodulin tested on their ability to antagonize CaM activity. Polymyxin B, ICso : 50% inhibitory concentration which is known to be a potent and selective antagonist of CaM [21], POE: cyclic 3': 5'-nucleotide phosphodiesterase was used as the control. Most drugs, even at high concentrations of

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343 344 HEGEMANN ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

Table II. Inhibition of Calmodulin (CaM)-Dependent and 100 6 -Independent Phosphodiesterase by Azoles Derivatives· 5 '0-... Phosphodiesterase c:: 80 -0 Drug CaM Dependent CaM Independent Selectivity u 0 60 Bifonazole 14.1 ± 2.7 380.0 ± 26.9 27.0 - Miconazole 11.3 ± 3.3 172.3 ± 18.6 15.3 ~0 Econazole 6.3 ± 1.9 208.3 ± 38.6 33.1 -c:: 40 Clotrimazole 18.4 ± 4.6 >1000.0' >54.3 4 0 Ketoconazole 65.2 ± 12.3 262 .5 ± 25 .3 4.0 .. 3 ;e • The values indicate the concentrations (in tiM) that induced 50% inhibition of .c: 20 enzyme activity (mean ± SEM in three to six independent experiments, each per­ c:: 2 formed in triplicate). The selectivity of these compounds towards the CaM-depen­ 1 dent isoenzyme, as compared to the CaM-independent isoenzyme, was calculated as 0 the ratio of the corresponding IC,. values. 9 8 765 4 3 • Higher concentrations of c1otrimazole were partially insoluble; 1 mM c1otrima­ zole induced inhibition of CaM-independent phosphodiesterase by 28.3 ± 2.4% of - Ig [drug] (M) the control.

Figure 1. Inhibition of calmodulin-dependent phosphodiesterase, used as an indicator enzyme for calmodulin activity, by various antifungal PDE was due to interference with CaM rather than by direct inhibi­ drugs: 1) 5-fluorocytosine, 2) amphotericin B, 3) nystatin, 4) tolnaftate, 5) clotrirnazole, 6) polymyxin B (used as a reference antagonist) [21] . The tion of the apoenzyme, PDE. However, the individual selectivity results shown are means of three to five independent experiments, each was found to vary between the different drugs. Clotrimazole dis­ performed in triplicate. played the highest selectivity of more than 55 times, whereas ketoconazole was the least selective drug, being only about four times more potent on CaM-dependent than on CaM-independent 1 mM, only marginally inhibited CaM-dependent PDE (Fig 1, PDE. Table I), which was used as an indicator enzyme for CaM activity. The interaction of the azole derivatives with CaM was further However, the azole derivative clotrimazole effectively inhibited investigated by performing dose-response curves in the presence of CaM-dependent PDE in a dose-dependent manner, displaying a increasing concentrations of CaM (10, 200, or 500 ng) for each half-maximal inhibition at a drug concentration (50% inhibitory azole derivative. As shown in Fig 3 for bifonazole, enzyme inhibi­ concentration (ICso) value) of 18.4 ± 4.6,uM (n = 5) . Similar re­ tion was reversed by increasing concentrations of CaM present in sults were obtained with all azole derivatives (Table II), which were the assay, as reflected by the characteristic shift of the dose-response found to inhibit CaM-dependent PDE in a dose-dependent manner curve to the right. Similar results were obtained with all azole deriv­ (Table I) with ICso values in the low micromolar range. The least atives, indicating a competitive interaction of these drugs with effective derivative was ketoconazole (ICso value, 65.2 ± 12.3 ,uM; CaM. This finding was further supported by a kinetic analysis of the n = 4). inhibition of CaM-dependent PDE by bifonazole using a double­ Inhibition of CaM-dependent PDE could be based on two dif­ reciprocal Lineweaver-Burk plot (Fig 4). This plot demonstrates ferent modes of action, direct antagonism of CaM or interference that the maximum enzyme activity remained unaltered in the pres­ with the detection enzyme, PDE, particularly with its binding site ence of the drug, whereas higher concentrations of CaM were neces­ for the substrate, adenosine 3' : 5' -cyclic monophosphate. To inves­ sary to stimulate the enzyme. This finding characterizes the drug as tigate these two possibilities, we compared the effects of each azole a competitive inhibitor of CaM. derivative on the CaM-independent and CaM-dependent isoen­ zyme from the same donor tissue. As shown for bifonazole (Fig 2), DISCUSSION the drug inhibited both isoenzymes but much higher concentra­ tions were required to inhibit CaM-independent PDE to the same In the present study, we have investigated the ability of various extent as CaM-dependent PDE, which was reflected by the differ­ structurally unrelated drugs to antagonize CaM activity. The azole ent ICso values for both enzymes, 380.0 ± 26.9 ,uM (n = 5) and derivates, bifonazole, econazole, miconazole, clotrimazole, and ke­ 14.1 ± 2.7 ,uM (n = 5), respectively. A comparable selectivity for toconazole, were all found to inhibit CaM in a dose-dependent CaM-dependent PDE was observed with all azole derivatives (Table manner. Inhibition of CaM-dependent PDE was selective with re­ II), indicating that drug-induced inhibition of CaM-dependent spect to the CaM-independent isoenzyme and competitive versus

Table I. Effects of Various Antifungal Drugs on Calmodulin Activity· Drug 1 JiM 10 JiM 100 JiM 1 mM Ciclopiroxolarnine 2.16 ± 0.02 2.07 ± 0.07 1.94 ± 0.15 1.04 ± 0.14 Nystatin 2.16 ± 0.01 1.99 ± 0.10 1.67 ± 0.05 1.41 ± 0.22 Amphotericin B 2.15 ± 0.02 2.13 ± 0.03 2.04 ± 0.03 1.96 ± 0.12 5-fluorocytosine 2.16 ± 0.02 2.07 ± 0.05 2.01 ± 0.04 2.02 ± 0.09 Tolnaftate 2.01 ± 0.04 1.99 ± 0.03 2.00 ± 0.05 1.41 ± 0.12 Griseofulvin 2.14 ± 0.02 2.07 ± 0.06 1.96 ± 0.06 1.44 ± 0.08 Bifonazole 2.01 ± 0.08 0.84 ± 0.05 0.07 ± 0.04 0.00 ± 0.01 Miconazole 1.91 ± 0.05 0.82 ± 0.04 0.29 ± 0.04 0.05 ± 0.02 Econazole 1.77 ± 0.04 0.68 ± 0.07 0.17 ± 0.05 0.08 ± 0.02 Clotrirnazole 1.96 ± 0.03 0.89 ± 0.18 0.39 ± 0.04 0.16 ± 0.09 Ketoconazole 2.09 ± 0.03 1.63 ± 0.06 0.88 ± 0.04 0.30 ± 0.02 • CaM activity, measured in the presence of various drug concentrations, was detected by its ability to stimulate CaM-dependent phosphodiesterase. The values indicate the phosphodiesterase activity (mean ± SEM from three to five independent experiments, each performed in triplicate) expressed as nmol adenosine 3' : 5' -cyclic monophosphate (cAMP) hydrolysed/min/mg enzyme. In the absence of drugs, the enzyme activity was 2.18 ± 0.24 nmol cAMP/min/mg. VOL. 100, NO.3 MARCH 1993 INHIBITION OF CALMODULIN BY AZOLES 345

100 -'0 ~ c -0 80 u 0 - 60 :::e0 -c 0 40 ;:: :g :c 20 .E 0 0 0 0.05 0.10 7 e 5 4 3 - Ig [blfonazole] (M) l/s Figure 4. Competitive interaction of bifonazole with calmodulin. Kinetic Figure 2. Inhibition of calmodulin-dependent (e) and -independent (II) phosphodiesterase by bifonazole, a representative azole derivative. analysis (Linewea,:,er-Burk ,Plot) of data (means of triplicate) obtained fr?m a .representative expenment (n = 3). The lili es represent values, ob­ Values are means SEM from five independent experiments, each per­ ± tamed m the presence of 0 (_), 10 (e), or 30 (A) j.lM bifonazC?le. formed in triplicate. The absence of error bars indicates that the SEM was smaller than the symbol.

study, might potentially contribute to their antifungal activity CaM. In contrast, the non-azole antifungal drugs tested, amphoter­ towards a broad spectrum of fungi. icin B, tolnaftate, griseofulvin, 5-fluorocytosine, nystatin, and ci­ . Secondly, the azole derivatives, particularly ketoconazole and clopiroxolamine, did not express significant inhibitory activity. ?Ifonazole, have .bee~ reported to be of therapeutic value in various The finding that the azole derivatives are competitive antagonists 1I1flammatory sk1l1 diseases. In clinical trials, bifonazole has been of CaM appears to be relevant to their clinical efficacy in two differ­ shown to be effective in the treatment of sebopsoriasis and rosacea ent contexts. Firstly, recent reports have suggested that CaM plays a [10]. Furthermore, bifonazo!e [3,6,9,10] and ketoconazole [4,5,7,8] regulatory role in the pathogenesis of infections by C. albicalls. CaM have successfully been used 111 the treatment of seborrheic dermati­ has been partially purified from this yeast [23] and well character­ tis. The common feature of these diseases is inflammation. In addi­ ized CaM antagonists, trifluoperazine [18,19] and R 24571 [19]' tion, sebopsoriasis and seborrheic dermatitis are characterized by have been found to inhibit yeast-mycelial transition, a critical step in hyperproliferatlOn of the epidermis. The numerous cellular func­ the mechanism of infections by C. albicalls [20]. Although the azole tions,. in which CaM. has ~een demonstrated to playa regulatory derivatives are believed to exert their antifungal efficacy by inhibi­ role, 1I1clude cell proliferation a.nd inflammatory responses (for re­ tion of ergosterol biosynthesis, thus interfering with the membrane vle~, s~e [15]~ . There~or e, the direct inhibition of CaM by the azole formation of fungi [12], the antagonism of CaM, as reported in this denvatlv~s ml ~ ht be 111 part the mechanism by which these drugs exert their antl-1I1flammatory activity. The drug concentrations applied clinically, i.e., 1 % bifonazole and 2% ket?conazole, equal a~out 32 mM and 38 mM, respec­ t~vely . CO~1S1denng the high sk1l1 penetration of the azole deriva­ tives, speclfical.ly ofblfonazole (for review, see [2]), and a life-time of t~ese dru~s 111 the skin ~f more than 30 h [20J , the drug concen­ 100 -'0 ~rat~ons achieve? aft~r t~plcal application appear to be sufficient to ~ 1I1hlblt CaM activity m IJ/IJO. The potencies of the azole derivatives as C - C~ antag~nists are comparable to those of the naphthalenesulfon­ 0 80 u amldes~ wh~ch. are "classical" CaM antagonists [24], and to that of 0 the antlpsonatlc d~g, anthralin [25J. In conclusion, the present data - 80 :::e raise the hypotheS IS that direct 1l1teractions of azole derivatives with ~ C CaM might contribute to the anti-inflammatory and antifungal 0 40 activity of these drugs. ;e:;: ~ 20 .= The expert teclltJi ca ~ assistallce oJMs. Christille L. Haml is gratifully ackllowledged . Th ese sflldl es were ltJ part supported by the Dermatology FOlmda tiotl. 0 7 6 5 4 3 - Ig [blfonazole] (M) REFERENCES Figure 3. Dose-response curve of bifonazole on calmodulin-dependent phosphodiesterase in the presence of various concentrations of calmodu­ 1. Plempel M: Experiences, recognitions and questions in azole antimy­ lin (A, 10; e, 200; _, 500 ng per 500 III assay volume). The data concs. Jpn J Med Mycol 23:17 -27, 1982 (means ± SD of triplicate assays) are derived from a representative exper­ 2. Lackner TE, Clissold SP: Bifonazole: a review of its antimicrobial iment (n = 3). The absence of error bars inclicates that the SD was smaller activity and therapeutic lise in superficial mycoses. Drugs 38:204- than the symbol. Similar results were obtained with other azole deriva­ 225, 1989 tives tested. 3. Roberts DT, Richardson MD, Main RA, Mann TS: Bifonazole gel in 346 HEGEMANN ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

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