Target Abundance-Based Fitness Screening (TAFiS) Facilitates Rapid Identification of Target-Specific and Physiologically Active Chemical Probes.
Target Abundance-Based Fitness Screening (TAFiS) Facilitates Rapid Identification of Target-Specific and Physiologically Active Chemical Probes.
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DOI：
10.1128/msphere.00379-17
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发表时间：
2017-09
期刊：
影响因子：
4.8
通讯作者：
Palmer GE
中科院分区：
文献类型：
--
作者：
Butts A;DeJarnette C;Peters TL;Parker JE;Kerns ME;Eberle KE;Kelly SL;Palmer GE
传统的药物筛选通常采用基于靶点或基于细胞的方法。第一组依靠生化分析来检测纯化靶标的调节剂。然而，HITS往往缺乏药物样特性，如膜通透性和靶向性。基于细胞的筛选识别出诱导所需表型的化合物，但靶点尚不清楚，这严重限制了进一步的开发和优化。为了解决这些问题，我们开发了第二代基于靶点的全细胞筛选方法，该方法结合了化学遗传学和竞争适应度的原理，使得能够从单一筛选中识别靶标特异性和生理活性化合物。我们选择使用重要的人类真菌病原体白色念珠菌来验证这一方法，目的是为了寻找新的抗真菌靶标。然而，这种方法是广泛适用的，预计将大大减少从筛选Hit到先导化合物所需的时间和资源。传统的药物发现方法效率低得令人沮丧，而且有几个关键的限制，严重制约了我们快速识别和开发新的实验疗法的能力。为了解决这一问题，我们设计了一种基于竞争适应度原理的第二代靶标全细胞筛选方法，它可以快速识别靶标特异性和生理活性化合物。简而言之，构建表达预选目标蛋白的高、中、低水平的菌株，用光谱不同的荧光蛋白(FP)标记，并汇集。然后将汇集的菌株在各种小分子存在的情况下生长，并通过测量相应的FP标签的强度来比较混合培养中每个菌株的相对生长情况。化学诱导的群体迁移表明，小分子的生物活性依赖于目标蛋白的丰度，从而建立了特定的功能相互作用。在这里，我们描述了在流行的人类真菌病原体白色念珠菌中应用这项技术所需的分子工具，并使用两个具有良好特性的药物靶标-羊毛甾醇脱甲基酶和二氢叶酸还原酶来验证该方法。然而，我们的方法，我们称之为基于靶标丰度的适合度筛选(TAFiS)，应该适用于广泛的分子靶标，基本上适用于任何遗传上可驯化的微生物。重要性传统药物筛选通常采用基于靶点或基于细胞的方法。第一组依靠生化分析来检测纯化靶标的调节剂。然而，HITS往往缺乏药物样特性，如膜通透性和靶向性。基于细胞的筛选识别出诱导所需表型的化合物，但靶点尚不清楚，这严重限制了进一步的开发和优化。为了解决这些问题，我们开发了第二代基于靶点的全细胞筛选方法，该方法结合了化学遗传学和竞争适应度的原理，使得能够从单一筛选中识别靶标特异性和生理活性化合物。我们选择使用重要的人类真菌病原体白色念珠菌来验证这一方法，目的是为了寻找新的抗真菌靶标。然而，这种方法是广泛适用的，预计将大大减少从筛选Hit到先导化合物所需的时间和资源。
Conventional drug screening typically employs either target-based or cell-based approaches. The first group rely on biochemical assays to detect modulators of a purified target. However, hits frequently lack drug-like characteristics such as membrane permeability and target specificity. Cell-based screens identify compounds that induce a desired phenotype, but the target is unknown, which severely restricts further development and optimization. To address these issues, we have developed a second-generation target-based whole-cell screening approach that incorporates the principles of both chemical genetics and competitive fitness, which enables the identification of target-specific and physiologically active compounds from a single screen. We have chosen to validate this approach using the important human fungal pathogen Candida albicans with the intention of pursuing novel antifungal targets. However, this approach is broadly applicable and is expected to dramatically reduce the time and resources required to progress from screening hit to lead compound. Traditional approaches to drug discovery are frustratingly inefficient and have several key limitations that severely constrain our capacity to rapidly identify and develop novel experimental therapeutics. To address this, we have devised a second-generation target-based whole-cell screening assay based on the principles of competitive fitness, which can rapidly identify target-specific and physiologically active compounds. Briefly, strains expressing high, intermediate, and low levels of a preselected target protein are constructed, tagged with spectrally distinct fluorescent proteins (FPs), and pooled. The pooled strains are then grown in the presence of various small molecules, and the relative growth of each strain within the mixed culture is compared by measuring the intensity of the corresponding FP tags. Chemical-induced population shifts indicate that the bioactivity of a small molecule is dependent upon the target protein’s abundance and thus establish a specific functional interaction. Here, we describe the molecular tools required to apply this technique in the prevalent human fungal pathogen Candida albicans and validate the approach using two well-characterized drug targets—lanosterol demethylase and dihydrofolate reductase. However, our approach, which we have termed target abundance-based fitness screening (TAFiS), should be applicable to a wide array of molecular targets and in essentially any genetically tractable microbe. IMPORTANCE Conventional drug screening typically employs either target-based or cell-based approaches. The first group relies on biochemical assays to detect modulators of a purified target. However, hits frequently lack drug-like characteristics such as membrane permeability and target specificity. Cell-based screens identify compounds that induce a desired phenotype, but the target is unknown, which severely restricts further development and optimization. To address these issues, we have developed a second-generation target-based whole-cell screening approach that incorporates the principles of both chemical genetics and competitive fitness, which enables the identification of target-specific and physiologically active compounds from a single screen. We have chosen to validate this approach using the important human fungal pathogen Candida albicans with the intention of pursuing novel antifungal targets. However, this approach is broadly applicable and is expected to dramatically reduce the time and resources required to progress from screening hit to lead compound.