Estrogen Receptor Antagonists Are Anti-Cryptococcal Agents That Directly Bind EF Hand Proteins and Synergize with Fluconazole <italic toggle="yes">In Vivo</italic>

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 antifun...

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Autores principales: 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
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Publicado: American Society for Microbiology 2014
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spelling oai:doaj.org-article:a47f62ddda0341948a47a260086195b22021-11-15T15:45:11ZEstrogen Receptor Antagonists Are Anti-Cryptococcal Agents That Directly Bind EF Hand Proteins and Synergize with Fluconazole <italic toggle="yes">In Vivo</italic>10.1128/mBio.00765-132150-7511https://doaj.org/article/a47f62ddda0341948a47a260086195b22014-02-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.00765-13https://doaj.org/toc/2150-7511ABSTRACT 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.Arielle ButtsKristy KoselnyYeissa Chabrier-RosellóCamile P. SemighiniJessica C. S. BrownXuying WangSivakumar AnnaduraiLouis DiDoneJulie TabroffWayne E. ChildersMagid Abou-GharbiaMelanie WellingtonMaria E. CardenasHiten D. MadhaniJoseph HeitmanDamian J. KrysanAmerican Society for MicrobiologyarticleMicrobiologyQR1-502ENmBio, Vol 5, Iss 1 (2014)
institution DOAJ
collection DOAJ
language EN
topic Microbiology
QR1-502
spellingShingle Microbiology
QR1-502
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
Estrogen Receptor Antagonists Are Anti-Cryptococcal Agents That Directly Bind EF Hand Proteins and Synergize with Fluconazole <italic toggle="yes">In Vivo</italic>
description 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. 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.
format article
author 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
author_facet 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
author_sort Arielle Butts
title Estrogen Receptor Antagonists Are Anti-Cryptococcal Agents That Directly Bind EF Hand Proteins and Synergize with Fluconazole <italic toggle="yes">In Vivo</italic>
title_short Estrogen Receptor Antagonists Are Anti-Cryptococcal Agents That Directly Bind EF Hand Proteins and Synergize with Fluconazole <italic toggle="yes">In Vivo</italic>
title_full Estrogen Receptor Antagonists Are Anti-Cryptococcal Agents That Directly Bind EF Hand Proteins and Synergize with Fluconazole <italic toggle="yes">In Vivo</italic>
title_fullStr Estrogen Receptor Antagonists Are Anti-Cryptococcal Agents That Directly Bind EF Hand Proteins and Synergize with Fluconazole <italic toggle="yes">In Vivo</italic>
title_full_unstemmed Estrogen Receptor Antagonists Are Anti-Cryptococcal Agents That Directly Bind EF Hand Proteins and Synergize with Fluconazole <italic toggle="yes">In Vivo</italic>
title_sort estrogen receptor antagonists are anti-cryptococcal agents that directly bind ef hand proteins and synergize with fluconazole <italic toggle="yes">in vivo</italic>
publisher American Society for Microbiology
publishDate 2014
url https://doaj.org/article/a47f62ddda0341948a47a260086195b2
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