一种使用 NanoBRET 高通量筛选 PROTAC 细胞内靶标结合和细胞通透性的方法。
A High-Throughput Method to Prioritize PROTAC Intracellular Target Engagement and Cell Permeability Using NanoBRET.
机构信息
Promega Corporation, Fitchburg, WI, USA.
出版信息
Methods Mol Biol. 2021;2365:265-282. doi: 10.1007/978-1-0716-1665-9_14.
Target engagement and cell permeation are important parameters that may limit the efficacy of proteolysis-targeting chimeras (PROTACs). Here, we present an approach that facilitates both the quantitation of PROTAC binding affinity for an E3 ligase of interest, as well as the assessment of relative intracellular availability. We present a panel of E3 ligase target engagement assays based upon the NanoBRET Target Engagement platform. Querying E3 ligase engagement under live-cell and permeabilized-cell conditions allow calculation of an availability index that can be used to rank order the intracellular availability of PROTACs. Here we present examples where the cellular availability of PROTACs and their monovalent precursors are prioritized using NanoBRET assays for CRBN or VHL E3 ligases.
靶标结合和细胞通透性是可能限制蛋白水解靶向嵌合体（PROTAC）疗效的重要参数。在这里，我们提出了一种方法，既可以定量测定 PROTAC 与感兴趣的 E3 连接酶的结合亲和力，也可以评估相对细胞内可用性。我们提出了一组基于 NanoBRET 靶标结合平台的 E3 连接酶靶标结合测定。在活细胞和透化细胞条件下查询 E3 连接酶结合情况，可以计算出可用性指数，可用于对 PROTAC 的细胞内可用性进行排序。在这里，我们提供了一些例子，使用针对 CRBN 或 VHL E3 连接酶的 NanoBRET 测定法，对 PROTAC 及其单价前体的细胞可用性进行了优先级排序。
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