Contemporary drug discovery typically quantifies the effect of a molecule on a biological target using the equilibrium-derived measurements of IC₅₀, EC₅₀, or K_D. Kinetic descriptors of drug binding are frequently linked with the effectiveness of a molecule in modulating a disease phenotype; however, these parameters are yet to be fully adopted in early drug discovery. Nanoluciferase bioluminescence resonance energy transfer (NanoBRET) can be used to measure interactions between fluorophore-conjugated probes and luciferase fused target proteins. Here, we describe an intracellular NanoBRET competition assay that can be used to quantify cellular kinetic rates of compound binding to nanoluciferase-fused bromodomain and extra-terminal (BET) proteins. Comparative rates are generated using a cell-free NanoBRET assay and by utilizing orthogonal recombinant protein-based methodologies. A screen of known pan-BET inhibitors is used to demonstrate the value of this approach in the investigation of kinetic selectivity between closely related proteins.

当代药物发现通常使用 IC ₅₀ 、 EC ₅₀或K _D的平衡导出测量来量化分子对生物靶标的影响。药物结合的动力学描述符通常与分子调节疾病表型的有效性相关。然而，这些参数尚未在早期药物发现中得到充分采用。纳米荧光素酶生物发光共振能量转移 (NanoBRET) 可用于测量荧光团缀合探针与荧光素酶融合靶蛋白之间的相互作用。在这里，我们描述了一种细胞内 NanoBRET 竞争测定，可用于量化化合物与纳米荧光素酶融合的溴结构域和额外末端 (BET) 蛋白结合的细胞动力学速率。使用无细胞 NanoBRET 测定和基于正交重组蛋白的方法产生比较率。使用已知泛 BET 抑制剂的筛选来证明该方法在密切相关蛋白质之间的动力学选择性研究中的价值。