Antifungal Activity of Tamoxifen: In Vitro and In Vivo Activities and Mechanistic Characterization

Abstract

Tamoxifen (TAM), an estrogen receptor antagonist used primarily to treat breast cancer, has well-recognized antifungal properties, but the activity of TAM has not been fully characterized using standardized (i.e., CLSI) in vitro susceptibility testing, nor has it been demonstrated in an in vivo model of fungal infection. In addition, its mechanism of action remains to be clearly defined at the molecular level. Here, we report that TAM displays in vitro activity (MIC, 8 to 64 g/ml) against pathogenic yeasts (Candida albicans, other Candida spp., and Cryptococcus neoformans). In vivo, 200 mg/kg of body weight per day TAM reduced kidney fungal burden (؊1.5 log 10 CFU per g tissue; P ‫؍‬ 0.008) in a murine model of disseminated candidiasis. TAM is a known inhibitor of mammalian calmodulin, and TAM-treated yeast show phenotypes consistent with decreased calmodulin function, including lysis, decreased new bud formation, disrupted actin polarization, and decreased germ tube formation. The overexpression of calmodulin suppresses TAM toxicity, hypofunctional calmodulin mutants are hypersensitive to TAM, and TAM interferes with the interaction between Myo2p and calmodulin, suggesting that TAM targets calmodulin as part of its mechanism of action. Taken together, these experiments indicate that the further study of compounds related to TAM as antifungal agents is warranted.

In Vitro and In Vivo Effects of Tamoxifen against Larval Stage Echinococcus granulosus

“…Different research on mammalian cells and yeast clearly showed that TAM targets endoplasmic reticulum calcium pumps, protein kinase C, and/or CaM. Consequently, this targeting induces an increase in intracellular calcium as a non-estrogen receptor-dependent effect (18). Our in vitro results on the TAM effects suggest a calcium cell deregulation in treated protoscoleces (Fig.…”

“…It has a wide range of effects on mammalian cell physiology, such as induction of intracellular calcium release, apoptosis, and autophagy. Moreover, it possesses antioxidant and antiangiogenesis properties (18). Consistent with this wide range of cellular effects, TAM has been shown to target mammalian proteins such as calmodulin (CaM), protein kinase C, phospholipase C, phosphoinositide kinase, P-glycoprotein (Pgp), and swelling-induced chloride channels (19).…”

A Repurposing Approach Identifies Off-Patent Drugs with Fungicidal Cryptococcal Activity, a Common Structural Chemotype, and Pharmacological Properties Relevant to the Treatment of Cryptococcosis

“…Indeed, trifluoperazine, pimozide, and other antipsychotics are well-characterized calmodulin inhibitors (26). Recently, we confirmed that tamoxifen targets calmodulin as part of its antifungal activity against S. cerevisiae (27). As detailed below, we have shown that a number of these molecules directly bind C. neoformans calmodulin, suggesting that this activity may contribute to the mechanism of their antifungal properties.…”

“…Both of these approaches suggested that many of our hits may target calmodulin as part of their mechanism of action. Supporting that hypothesis was the fact that phenothiazines such as trifluoperazine, thioridazine, and prochlorpromazine are well-characterized, canonical calmodulin antagonists (26); similarly, tamoxifen, bepridil, and amiodarone have all been shown to inhibit mammalian calmodulin (27). Curiously, except for our previous studies supporting calmodulin as a target of tamoxifen in S. cerevisiae, the anticalmodulin properties of these drugs have not previously been invoked as a potential mechanism for their antifungal activity (27).…”

“…Supporting that hypothesis was the fact that phenothiazines such as trifluoperazine, thioridazine, and prochlorpromazine are well-characterized, canonical calmodulin antagonists (26); similarly, tamoxifen, bepridil, and amiodarone have all been shown to inhibit mammalian calmodulin (27). Curiously, except for our previous studies supporting calmodulin as a target of tamoxifen in S. cerevisiae, the anticalmodulin properties of these drugs have not previously been invoked as a potential mechanism for their antifungal activity (27). We confirmed that a significant number of the drugs identified in our screen bind directly to C. neoformans Cam1p in vitro and thus, for the first time, provide experimental support for a specific fungal protein target for drugs such as trifluoperazine, thioridazine, and fluspirilen.…”