Estrogen Receptor Antagonists Are Anti-Cryptococcal Agents That Directly Bind EF Hand Proteins and Synergize with Fluconazole In Vivo

Abstract

Cryptococcosis is an infectious disease of global significance for which new therapies are needed. Repurposing previously developed drugs for new indications can expedite the translation of new therapies from bench to beside. Here, we characterized the anti-cryptococcal activity and antifungal mechanism of estrogen receptor antagonists related to the breast cancer drugs tamoxifen and toremifene. Tamoxifen and toremifene are fungicidal and synergize with fluconazole and amphotericin B in vitro. In a mouse model of disseminated cryptococcosis, tamoxifen at concentrations achievable in humans combines with fluconazole to decrease brain burden by ~1 log10. In addition, these drugs inhibit the growth of Cryptococcus neoformans within macrophages, a niche not accessible by current antifungal drugs. Toremifene and tamoxifen directly bind to the essential EF hand protein calmodulin, as determined by thermal shift assays with purified C. neoformans calmodulin (Cam1), prevent Cam1 from binding to its well-characterized substrate calcineurin (Cna1), and block Cna1 activation. In whole cells, toremifene and tamoxifen block the calcineurin-dependent nuclear localization of the transcription factor Crz1. A large-scale chemical genetic screen with a library of C. neoformans deletion mutants identified a second EF hand-containing protein, which we have named calmodulin-like protein 1 (CNAG_05655), as a potential target, and further analysis showed that toremifene directly binds Cml1 and modulates its ability to bind and activate Cna1. Importantly, tamoxifen analogs (idoxifene and methylene-idoxifene) with increased calmodulin antagonism display improved anti-cryptococcal activity, indicating that calmodulin inhibition can be used to guide a systematic optimization of the anti-cryptococcal activity of the triphenylethylene scaffold.

Oral Administration of the Broad-Spectrum Antibiofilm Compound Toremifene Inhibits Candida albicans and Staphylococcus aureus Biofilm Formation In Vivo

“…In view of the inhibitory activity of toremifene against the fungus C. neoformans and the Ebola virus reported previously (17,28), our data further highlight the broad-spectrum antibiofilm activity of toremifene.…”

“…We already showed that the antibiofilm activity of toremifene against C. albicans is partly mediated by membrane permeabilization (15). Butts and colleagues further demonstrated that the fungicidal activity of toremifene against Cryptococcus neoformans is based on inhibition of calcineurin signaling via calmodulin binding (17). The findings of these two studies regarding the antifungal/antibiofilm mode of action of toremifene are in line with the well documented antifungal mechanism of action of its close analogue tamoxifen, which is based on membrane perturbation and interference with calcium homeostasis and calcineurin signaling (18)(19)(20).…”

Components of the Calcium-Calcineurin Signaling Pathway in Fungal Cells and Their Potential as Antifungal Targets

“…The results demonstrated that calmodulin mutants showed increased sensitivity to miconazole, terbinafine, or itraconazole compared with the sensitivities of the parent strains. Recently, another report demonstrated that the estrogen receptor antagonists tamoxifen and toremifene exerted their anticryptococcal activity alone or in combination with fluconazole and amphotericin B by directly binding to the essential EF-hand protein calmodulin, which prevented calmodulin from binding to its wellcharacterized substrate calcineurin and blocked calcineurin activation (110). This finding indicated that calmodulin antagonism contributes to the antifungal activity of this scaffold.…”

Drug repurposing screens and synergistic drug‐combinations for infectious diseases

“…Additionally, loperamide, a diarrhoea drug, was repurposed against Salmonella enterica (Ejim et al , 2011). The breast cancer drug tamoxifen showed efficacy in a murine model of cryptococcosis (Butts et al , 2014). Chlorcyclizine, an old antihistamine, was repurposed for the treatment of infections by the hepatitis C virus (He et al , 2015).…”