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

Arielle Butts; Kristy Koselny; Yeissa Chabrier-Roselló; Camile P Semighini; Jessica C. S Brown; Xuying Wang; Sivakumar Annadurai; Louis DiDone; Julie Tabroff; Wayne E Childers; Magid Abou-Gharbia; Melanie Wellington; Maria E Cardenas; Hiten D Madhani; Joseph Heitman; Damian J Krysan

doi:10.1128/mBio.00765-13

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

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

Arielle Butts, Kristy Koselny, Yeissa Chabrier-Roselló, Camile P Semighini, Jessica C. S Brown, Xuying Wang, Sivakumar Annadurai, Louis DiDone, Julie Tabroff, Wayne E Childers, …

mBio, Vol.5(1), pp.e00765-e00713

02/28/2014

DOI: 10.1128/mBio.00765-13

PMCID: PMC3950514

PMID: 24520056

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Abstract

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 log 10 . 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. 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.

Details

Title: Subtitle: Estrogen Receptor Antagonists Are Anti-Cryptococcal Agents That Directly Bind EF Hand Proteins and Synergize with Fluconazole In Vivo
Creators: Arielle Butts - Department of Chemistry, University of Rochester, Rochester, New York, USA
Kristy Koselny - Department of Pediatrics, University of Rochester, Rochester, New York, USA
Yeissa Chabrier-Roselló - Department of Molecular Genetics and Microbiology, Duke University, Durham, North Carolina, USA
Camile P Semighini - Department of Molecular Genetics and Microbiology, Duke University, Durham, North Carolina, USA
Jessica C. S Brown - Department of Biochemistry and Biophysics, University of California-San Francisco, San Francisco, California, USA
Xuying Wang - Department of Molecular Genetics and Microbiology, Duke University, Durham, North Carolina, USA
Sivakumar Annadurai - Moulder Center for Drug Discovery Research, Temple University School of Pharmacy, Philadelphia, Pennsylvania, USA
Louis DiDone - Department of Pediatrics, University of Rochester, Rochester, New York, USA
Julie Tabroff - Department of Chemistry, University of Rochester, Rochester, New York, USA
Wayne E Childers - Moulder Center for Drug Discovery Research, Temple University School of Pharmacy, Philadelphia, Pennsylvania, USA
Magid Abou-Gharbia - Moulder Center for Drug Discovery Research, Temple University School of Pharmacy, Philadelphia, Pennsylvania, USA
Melanie Wellington - Department of Pediatrics, University of Rochester, Rochester, New York, USA
Maria E Cardenas - Department of Molecular Genetics and Microbiology, Duke University, Durham, North Carolina, USA
Hiten D Madhani - Department of Biochemistry and Biophysics, University of California-San Francisco, San Francisco, California, USA
Joseph Heitman - Department of Molecular Genetics and Microbiology, Duke University, Durham, North Carolina, USA
Damian J Krysan - Department of Pediatrics, University of Rochester, Rochester, New York, USA, Department of Microbiology/Immunology, University of Rochester, Rochester, New York, USA
Contributors: Liise-anne Pirofski (Editor)
Resource Type: Journal article
Publication Details: mBio, Vol.5(1), pp.e00765-e00713
DOI: 10.1128/mBio.00765-13
PMID: 24520056
PMCID: PMC3950514
NLM abbreviation: mBio
ISSN: 2161-2129
eISSN: 2150-7511
Language: English
Date published: 02/28/2014
Academic Unit: Microbiology and Immunology; Stead Family Department of Pediatrics; Infectious Disease (Pediatrics)
Record Identifier: 9984093372802771

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