Identification of ligand binding by protein stabilization: Comparison of ATLAS with biophysical and enzymatic methods
Identification of ligand binding by protein stabilization: Comparison of ATLAS with biophysical and enzymatic methods
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DOI：
10.1089/adt.2007.100
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发表时间：
2008-02-01
影响因子：
1.8
通讯作者：
Appleman, James R.
中科院分区：
文献类型：
--
作者：
Thompson, Peggy A.;Wang, Shaohui;Appleman, James R.
Atlas(TM)(Any Target Ligand Affity Screen)(Anadys PharmPharmticals，Inc.，San Diego，CA)是一种基于蛋白质热变性和配体结合并稳定目标蛋白质的能力的同质、基于亲和力的高通量筛选技术。为了进一步了解识别与可溶性蛋白靶标结合的配体的检测灵敏度，我们选择了萤火虫荧光素酶来表征这项技术。荧光素酶是一种多结构域蛋白质，具有复杂的去折叠途径。三磷酸腺苷的结合导致结构域的稳定构象重排。通过使用荧光素酶对ATLAS技术进行表征，我们可以评估用于鉴定与任何靶标结合的配体的筛选方法的通用性。从功能筛选中鉴定的荧光素酶抑制剂被用来评估ATLAS对顺序抑制剂进行排序的能力。ATLAS 50%有效浓度与其他生物物理和生化方法的比较有助于优化ATLAS检测条件，以最大限度地提高化合物结合和蛋白质稳定性的灵敏度。结果表明，表征蛋白质的热展开和聚集行为对于优化ATLAS筛选具有重要意义。
ATLAS (TM) (Any Target Ligand Affinity Screen) (Anadys Pharmaceuticals, Inc., San Diego, CA) is a homogeneous, affinity-based high-throughput screening technology based on protein thermal denaturation and the ability of ligands to bind and stabilize the target protein from unfolding. To further understand the assay sensitivity for the identification of ligands that bind to soluble protein targets, firefly luciferase was chosen to characterize the technology. Luciferase is a multidomain protein with a complex unfolding pathway. Binding of ATP results in a stabilizing conformational rearrangement of the domains. Using luciferase to characterize the ATLAS technology allowed us to evaluate the generality of the screening method for the identification of ligand binding to any target. Luciferase inhibitors identified from functional screens were used to assess the capability of ATLAS to rank order inhibitors. Comparison of the ATLAS 50% effective concentration with other biophysical and biochemical methods offered insight into optimizing ATLAS assay conditions to maximize sensitivity to compound binding and protein stabilization. The results show the importance of characterizing the thermal unfolding and aggregation behavior of the protein to allow the ATLAS screen to be optimally designed.