A Bead-Based Proximity Assay for BRD4 Ligand Discovery.
A Bead-Based Proximity Assay for BRD4 Ligand Discovery.
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DOI：
10.1002/9780470559277.ch150024
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发表时间：
2015-12-02
影响因子：
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通讯作者：
Bradner JE
中科院分区：
文献类型：
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作者：
Roberts JM;Bradner JE
含溴结构域的蛋白质已成为抗肿瘤和抗炎药物发现的理想靶点。为了开发溴结构域BET家族的选择性抑制剂，我们优化了基于微球的检测方法，以检测溴结构域与多乙酰化组蛋白多肽之间的相互作用。通过这种蛋白质-蛋白质相互作用，结合到靶标和配体上的供体和受体微珠变得接近。激光照射后，施主微珠释放出的单线态氧进入空间位置较近的受主微珠，产生化学发光。这一AlphaScreen分析已被证明可用于高通量筛选、二次验证和在线索发现和优化过程中的特异性分析。在这里，我们报告了我们的检测开发方案，以衡量配体与含有溴域的蛋白4(BRD4)结合的抑制程度。我们讨论了合适的探针的发现，珠子、探针和蛋白质浓度的优化，以及蛋白质探针抑制曲线的推导。最后，我们探索了这项技术的实施，以高通量筛选潜在的BRD4抑制剂。
Bromodomain-containing proteins have emerged as desirable targets for anti-neoplastic and anti-inflammatory drug discovery. Toward the development of selective inhibitors of the BET family of bromodomains, we optimized bead-based assays to detect interactions between bromodomains and poly-acetylated histone peptides. Donor and acceptor beads bound to target and ligand are brought into proximity by this protein-protein interaction. After laser illumination, singlet oxygen evolved from donor beads travels to the spatially close acceptor beads, resulting in chemiluminesence. This AlphaScreen assay has proven amendable to high-throughput screening, secondary validation, and specificity profiling during lead discovery and optimization. Here we report our protocol for assay development to measure inhibition of ligand binding to bromodomain containing protein 4 (BRD4). We discuss the discovery of an appropriate probe, optimization of bead, probe, and protein concentrations, and the derivation of protein-probe inhibition curves. Finally, we explore the implementation of this technology for high-throughput screening of potential BRD4 inhibitors.