Roberts Justin M, Bradner James E
Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts.
Department of Medicine, Harvard Medical School, Boston, Massachusetts.
Curr Protoc Chem Biol. 2015 Dec 2;7(4):263-278. doi: 10.1002/9780470559277.ch150024.
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.
含溴结构域的蛋白质已成为抗肿瘤和抗炎药物研发的理想靶点。为了开发溴结构域BET家族的选择性抑制剂，我们优化了基于微珠的检测方法，以检测溴结构域与多乙酰化组蛋白肽之间的相互作用。通过这种蛋白质-蛋白质相互作用，与靶标和配体结合的供体微珠和受体微珠被拉近。激光照射后，从供体微珠产生的单线态氧扩散到空间上靠近的受体微珠，从而产生化学发光。这种AlphaScreen检测方法已被证明适用于先导化合物发现和优化过程中的高通量筛选、二次验证和特异性分析。在此，我们报告了用于检测配体与含溴结构域蛋白4（BRD4）结合抑制作用的检测方法开发方案。我们讨论了合适探针的发现、微珠、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，我们探索了该技术在高通量筛选潜在BRD4抑制剂中的应用。
