Ketoconazole binds to glucocorticoid receptors and exhibits glucocorticoid antagonist activity in cultured cells. D S Loose, … , E P Stover, D Feldman J Clin Invest. 1983;72(1):404-408. https://doi.org/10.1172/JCI110982. Research Article We have recently found that ketoconazole inhibits adrenal steroidogenesis; in this paper we investigated whether imidazole antimycotic drugs additionally interact with glucocorticoid receptor sites in target tissues. Our approach was to assess the ability of three drugs: ketoconazole, clotrimazole, and RS 49910, to inhibit [3H]dexamethasone binding to hepatoma tissue culture (HTC) cell cytosol. The results indicated dose-dependent, competitive displacement of [3H]dexamethasone binding that was in the potency sequence: clotrimazole greater than ketoconazole greater than RS 49910. We then examined the functional response of this binding by measuring tyrosine aminotransferase (TAT) activity in HTC cells. The antimycotics did not exhibit TAT agonist activity and inhibition of basal enzyme levels was not detected. However, the drugs were potent antagonists of dexamethasone-induced TAT activity and the effect was temporally reversible. This antagonist activity was in the same sequence and closely correlated with the binding potency of the three drugs. We conclude that ketoconazole and other imidazole antimycotic drugs possess glucocorticoid antagonist activity by virtue of occupancy of glucocorticoid receptor sites in target tissues. Find the latest version: https://jci.me/110982/pdf Ketoconazole Binds to Glucocorticoid Receptors and Exhibits Glucocorticoid Antagonist Activity in Cultured Cells DAVID S. LOOSE, E. PRICE STOVER, and DAVID FELDMAN, Stanford University School of Medicine, Department of Medicine, Stanford, California 94305 A B S T R A C T We have recently found that ketocon- onstrated that ketoconazole is a potent inhibitor of ste- azole inhibits adrenal steroidogenesis; in this paper we roidogenesis both in volunteers and in in vitro studies investigated whether imidazole antimycotic drugs ad- with adrenal (2, 3) and testicular cells (4). Because ditionally interact with glucocorticoid receptor sites other drugs known to inhibit steroidogenesis (5) also in target tissues. Our approach was to assess the ability compete for specific steroid receptor binding sites in of three drugs: ketoconazole, clotrimazole, and RS target tissues (6), we proceeded to examine the pos- 49910, to inhibit [3H]dexamethasone binding to hep- sibility that ketoconazole might additionally interact atoma tissue culture (HTC) cell cytosol. The results with glucocorticoid receptors. Since numerous other indicated dose-dependent, competitive displacement imidazole antimycotic drugs are available or are in of [3H]dexamethasone binding that was in the potency various stages of development (1), we also examined sequence: clotrimazole > ketoconazole > RS 49910. two additional structurally related drugs to ascertain We then examined the functional response of this whether any activity found for ketoconazole was spe- binding by measuring tyrosine aminotransferase (TAT) cific to that molecule or represented more general activity in HTC cells. The antimycotics did not exhibit properties associated with the imidazole antifungal TAT agonist activity and inhibition of basal enzyme drugs as a family. Two sets of experiments were un- levels was not detected. However, the drugs were po- dertaken. First, the antifungal agents were tested for tent antagonists of dexamethasone-induced TAT ac- their ability to compete with [3H]dexamethasone for tivity and the effect was temporally reversible. This glucocorticoid receptor binding sites in cytosol pre- antagonist activity was in the same sequence and pared from hepatoma tissue culture (HTC)' cells, a closely correlated with the binding potency of the classical glucocorticoid target system (7, 8). Second, three drugs. We conclude that ketoconazole and other the antifungal drugs were tested in HTC cells for their imidazole antimycotic drugs possess glucocorticoid ability to either induce or inhibit the glucocorticoid antagonist activity by virtue of occupancy of gluco- induction of the enzyme tyrosine aminotransferase corticoid receptor sites in target tissues. (TAT; 7, 8). The results of these studies indicate that ketoconazole, and the other related antifungal agerits, INTRODUCTION competitively inhibit [3H]dexamethasone binding to HTC cell glucocorticoid receptors and effectively Ketoconazole is an important new antifungal agent block dexamethasone induction of TAT. These find- because of its effectiveness against a wide variety of ings raise the possibility that this class of drugs may pathogenic fungi after oral administration and its lim- prove useful as glucocorticoid antagonists. ited number of side effects (1). However, recent in- vestigations in this and other laboratories have dem- METHODS Cells and tissue culture. HTC cells were routinely grown in T75 flasks in Dulbecco's Modified Eagle's Medium sup- Address all correspondence to Dr. Feldman. Received for publication 22 February 1983 and in revised ' Abbreviations used in this paper: HTC, hepatoma tissue form 6 April 1983. culture; TAT, tyrosine aminotransferase. 404 J. Clin. Invest. © The American Society for Clinical Investigation, Inc. * 0021-9738/83/07/0404/05 $1.00 Volume 72 July 1983 404-408 plemented with 5% newborn calf serum, 5% fetal calf serum (all from Gibco, Grand Island, NY), and 1 g/liter glucose. For binding studies, cells were grown for 7-8 d (confluence) with a medium change on days 2 and 5. For enzyme assays, o 4910 cells were grown for 4-5 d, with serum-free medium used for the last 24 h. oz 75 KETO Binding assays. Cytosol binding studies were performed using previously reported techniques (9). Briefly, cells were z at 0°C in a uw50-- harvested by scraping and lysed by sonication z medium containing 250 mM sucrose, 1.5 mM EDTA, 10 mM 0 Tris, 12 mM monothioglycerol, and 10 mM sodium molyb- ,, CLO < a date, pH 7.8. Cytosol was prepared by centrifugation at O 204,000 g for 30 min. Glucocorticoid receptors were assayed ui 25 by incubating aliquots of cytosol with [3H]dexamethasone x (26 Ci/mmol, Amersham Corp., Arlington Heights, IL) with 0 -v- .. or without competitors for 3 h at 0°C. A correction for non- 1 10 100 specific binding was made in all experiments by subtracting that binding resistant to a 250-fold molar excess of radioinert ANTIMYCOTIC DRUG CONCENTRATION (PM) dexamethasone. Bound hormone was separated from free FIGURE 1 Antifungal drug competition for specific hormone using mini-columns made with G-50 fine Sephadex [3H]dexamethasone binding sites in HTC cell cytosol. Cytosol (Pharmacia Fine Chemicals, Piscataway, NJ; 9). was incubated with 13 nM [3H]dexamethasone with or with- Tyrosine aminotransferase activity. TAT activity was out the indicated concentration of competitors for 3 h at assessed by a spectrophotometric method (7, 8). Cells were 0°C. Results are expressed as a percentage of control binding treated with various antifungal agents with or without dexa- obtained in the absence of competitors (109±14 fmol/mg methasone for 20 h, lysed by sonication, and aliquots of the protein). Values shown are mean±SE of four to six deter- 8,000-g supernatant were assayed. Soluble protein was de- minations. CLO, clotrimazole; KETO, ketoconazole, 49910, termined by the method of Bradford (10). Syntex drug RS 49910. Drugs. Ketoconazole was obtained from Janssen (Beerse, Belgium). Clotrimazole (11), a topical antifungal developed by Bayer, and RS 49910, an aryloxy-substituted alkylim- against [3H]estradiol binding to estrogen receptors in idazole (Syntex experimental antifungal), were both ob- tained from Syntex (Palo Alto, CA). Since circulating drug rat uterus (13) and against [3H]1,25(OH)2vitamin D3 levels are usually expressed on the basis of microgram per binding to vitamin D receptors in rat intestine (14). milliliter and all of our studies are based on molar concen- In both cases, 10 gg/ml of ketoconazole failed to sig- trations, we note here that 1.88 gM ketoconazole is equiv- nificantly compete for the binding sites indicating that alent to 1 Ag/ml. the action against the glucocorticoid receptor was re- ceptor-specific. Second, Lineweaver-Burk analysis of the ketoconazole competition for the glucocorticoid RESULTS receptor revealed that the inhibition of binding was Ability of antimycotics to compete for receptor competitive in nature. Third, these inhibitors were also binding sites. Our first experiments were designed examined against glucocorticoid receptors freshly pre- to ascertain whether antifungal drugs competed with pared from either kidney or thymus cytosol obtained [3H]dexamethasone for glucocorticoid receptor bind- from adrenalectomized rats (9). The results were es- ing sites. As shown in Fig. 1, each of the three drugs sentially the same as with HTC cell cytosol. inhibited [3H]dexamethasone binding to HTC cell cy- Effect of antimycotic drugs on TA T induction. tosol, but with a widely differing spectrum of activity. Occupancy of the glucocorticoid receptor may result The order of potency and the concentration inhibiting in either the expression of a glucocorticoid-induced 50% [3H]dexamethasone binding was: clotrimazole (0.7 function (agonist activity) or blockade of a function AM or 0.24 jug/ml) > ketoconazole (20 MM or 10 ,g/ induced by glucocorticoids (antagonist activity). To ml) > RS-49910 (50 MM or 21 Ag/ml). To put these determine which of these possibilities resulted from