Butts Arielle, DeJarnette Christian, Peters Tracy L, Parker Josie E, Kerns Morgan E, Eberle Karen E, Kelly Steve L, Palmer Glen E
Department of Clinical Pharmacy and Translational Science, College of Pharmacy, University of Tennessee Health Sciences Center, Memphis, Tennessee, USA.
Department of Molecular Immunology and Biochemistry, College of Graduate Health Sciences, University of Tennessee Health Sciences Center, Memphis, Tennessee, USA.
mSphere. 2017 Oct 4;2(5). doi: 10.1128/mSphere.00379-17. eCollection 2017 Sep-Oct.
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 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. 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 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.
传统的药物研发方法效率极低，令人沮丧，且存在几个关键局限性，严重限制了我们快速识别和开发新型实验性治疗药物的能力。为了解决这个问题，我们基于竞争适应性原理设计了一种第二代基于靶点的全细胞筛选测定法，该方法可以快速识别靶点特异性和生理活性化合物。简而言之，构建表达预选靶点蛋白高、中、低水平的菌株，用光谱不同的荧光蛋白（FPs）进行标记，然后混合。将混合菌株在各种小分子存在的情况下培养，通过测量相应FP标签的强度来比较混合培养物中每个菌株的相对生长情况。化学诱导的群体变化表明小分子的生物活性取决于靶点蛋白的丰度，从而建立特定的功能相互作用。在这里，我们描述了将该技术应用于常见的人类真菌病原体所需的分子工具，并使用两个特征明确的药物靶点——羊毛甾醇去甲基化酶和二氢叶酸还原酶验证了该方法。然而，我们的方法，即基于靶点丰度的适应性筛选（TAFiS），应该适用于广泛的分子靶点，并且基本上适用于任何遗传上易于处理的微生物。传统的药物筛选通常采用基于靶点或基于细胞的方法。第一类方法依赖于生化测定来检测纯化靶点的调节剂。然而，筛选出的化合物常常缺乏类似药物的特性，如膜通透性和靶点特异性。基于细胞的筛选可以识别诱导所需表型的化合物，但靶点未知，这严重限制了进一步的开发和优化。为了解决这些问题，我们开发了一种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性原理，能够从单次筛选中识别靶点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来验证这种方法，目的是寻找新的抗真菌靶点。然而，这种方法具有广泛的适用性，预计将大大减少从筛选出的命中化合物到先导化合物的研发所需的时间和资源。
