Estrogen receptor antagonists are anti-cryptococcal agents that directly bind EF hand proteins and synergize with fluconazole in vivo.
Estrogen receptor antagonists are anti-cryptococcal agents that directly bind EF hand proteins and synergize with fluconazole in vivo.
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DOI：
10.1128/mbio.00765-13
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发表时间：
2014-02-11
期刊：
影响因子：
6.4
通讯作者：
Krysan DJ
中科院分区：
文献类型：
--
作者：
Butts A;Koselny K;Chabrier-Roselló Y;Semighini CP;Brown JC;Wang X;Annadurai S;DiDone L;Tabroff J;Childers WE Jr;Abou-Gharbia M;Wellington M;Cardenas ME;Madhani HD;Heitman J;Krysan DJ
隐球菌病是一种具有全球意义的传染病，需要新的治疗方法。将以前开发的药物重新用于新的适应症可以加快新疗法从实验室到旁边的转变。在这里，我们描述了与乳腺癌药物他莫昔芬和托瑞米芬相关的雌激素受体拮抗剂的抗隐球菌活性和抗真菌机制。他莫昔芬和托瑞米芬在体外具有杀菌作用，并与氟康唑和阿替霉素B有协同作用。在播散性隐球菌病的小鼠模型中，在人体可达到的浓度下，他莫昔芬与氟康唑联合使用可使脑负荷降低约1 log 10。此外，这些药物抑制巨噬细胞内新生隐球菌的生长，这是目前抗真菌药物无法达到的利基。托瑞米芬和他莫昔芬直接结合必需的EF手蛋白钙调蛋白，如通过纯化的新型隐球菌钙调蛋白（Cam 1）的热位移测定所确定的，阻止Cam 1结合其充分表征的底物钙调磷酸酶（Cna 1），并阻断Cna 1活化。在全细胞中，托瑞米芬和他莫昔芬阻断了转录因子Crz 1依赖于钙调神经磷酸酶的核定位。利用新型隐球菌缺失突变体文库进行的大规模化学遗传筛选确定了第二种含EF手的蛋白质，我们将其命名为钙调蛋白样蛋白1（CNAG_05655），作为潜在靶点，进一步分析表明托瑞米芬直接结合Cml 1并调节其结合和激活Cna 1的能力。重要的是，具有增加的钙调蛋白拮抗作用的他莫昔芬类似物（idoxifene和亚甲基-idoxifene）显示出改善的抗隐球菌活性，表明钙调蛋白抑制可用于指导三苯乙烯支架的抗隐球菌活性的系统优化。在全世界，隐球菌病每年影响约100万人，每年杀死的艾滋病毒/艾滋病患者多于结核病。隐球菌病的金标准疗法是阿替霉素B加5-氟胞嘧啶，但在资源有限和疾病负担最高的地区，这种疗法并不容易获得。在本文中，我们表明，与乳腺癌药物他莫昔芬相关的分子是隐球菌属的杀真菌剂，并显示出许多抗隐球菌药物所需的药理学特性，包括与氟康唑在体外和体内的协同杀真菌活性，口服生物利用度和巨噬细胞内的活性。我们还证明了这类分子靶向钙调蛋白作为其作用机制的一部分，并且具有增加的钙调蛋白拮抗作用的他莫昔芬类似物具有改善的抗隐球菌活性。总之，这些结果表明，他莫昔芬是一个非常有吸引力的新的抗隐球菌药物的开发支架，并提供了其进一步优化的机制基础。
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. 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.