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.
IMPORTANCE: Worldwide,
cryptococcosis affects approximately 1 million people annually and kills more HIV/
AIDS patients per year than
tuberculosis. The gold standard
therapy for
cryptococcosis is
amphotericin B plus 5-flucytosine, but this regimen is not readily available in regions where resources are limited and where the burden of disease is highest. Herein, we show that molecules related to the
breast cancer drug tamoxifen are fungicidal for Cryptococcus and display a number of pharmacological properties desirable for an anti-cryptococcal
drug, including synergistic fungicidal activity with
fluconazole in vitro and in vivo, oral bioavailability, and activity within macrophages. We have also demonstrated that this class of molecules targets
calmodulin as part of their mechanism of action and that
tamoxifen analogs with increased
calmodulin antagonism have improved anti-cryptococcal activity. Taken together, these results indicate that
tamoxifen is a pharmacologically attractive scaffold for the development of new anti-cryptococcal drugs and provide a mechanistic basis for its further optimization.