Monroy Erika Y, Yu Xin, Lu Dong, Qi Xiaoli, Wang Jin
Verna and Marrs McLean Department of Biochemistry and Molecular Pharmacology, Baylor College of Medicine, Houston, Texas 77030, United States.
Center for NextGen Therapeutics, Baylor College of Medicine, Houston, Texas 77030, United States.
bioRxiv. 2025 Mar 27:2025.03.24.645143. doi: 10.1101/2025.03.24.645143.
Target engagement assays are critical for drug discovery, with Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) and Nano Bioluminescence Resonance Energy Transfer (NanoBRET) representing two complementary approaches for biochemical and cellular evaluation. Traditionally, these platforms demand distinct fluorescent tracers tailored to their unique detection systems, requiring separate probe development for comprehensive target characterization. Despite their widespread adoption, the development of platform-specific fluorescent tracers often leads to increased costs and experimental complexity. In this study, two fluorescent tracers, T2- BODIPY -FL and T2-BODIPY-589, initially developed for receptor-interacting protein kinase 1 (RIPK1) target engagement studies in TR-FRET and NanoBRET applications respectively, were systematically evaluated for their performance across both platforms under various detection parameters. By evaluating their performance across both assay systems, we demonstrate that both tracers can effectively bridge biochemical and cellular assays, delivering reliable measurements. T2-BODIPY-589, with its red-shifted spectral properties, exhibits superior performance in NanoBRET assays (Z' up to 0.80) while maintaining acceptable functionality in TR-FRET systems (Z'=0.53). In contrast, T2-BODIPY -FL provides optimal performance for TR-FRET (Z'=0.57) but also demonstrates potential for use in NanoBRET (Z' up to 0.72), albeit with reduced efficiency. Competition assays with an unlabeled inhibitor yielded consistent binding constants across all tracer-platform combinations, validating their reliability for quantitative measurements. Our findings highlight the potential for integrating a single tracer across diverse assay platforms, reducing the need for separate probe development and enhancing experimental consistency. This approach has broad implications for streamlining assay development, improving data comparability, and enables more direct comparisons between biochemical and cellular data, with broader implications for integrated drug discovery programs across diverse target classes.
靶点结合分析对于药物发现至关重要，时间分辨荧光共振能量转移（TR-FRET）和纳米生物发光共振能量转移（NanoBRET）是用于生化和细胞评估的两种互补方法。传统上，这些平台需要针对其独特检测系统定制的不同荧光示踪剂，需要单独开发探针以全面表征靶点。尽管它们被广泛采用，但特定平台荧光示踪剂的开发往往会导致成本增加和实验复杂性提高。在本研究中，两种荧光示踪剂T2-硼二吡咯-FL和T2-硼二吡咯-589，最初分别为TR-FRET和NanoBRET应用中的受体相互作用蛋白激酶1（RIPK1）靶点结合研究而开发，在各种检测参数下对其在两个平台上的性能进行了系统评估。通过评估它们在两种分析系统中的性能，我们证明这两种示踪剂都可以有效地连接生化和细胞分析，提供可靠的测量结果。T2-硼二吡咯-589具有红移光谱特性，在NanoBRET分析中表现出卓越性能（Z'高达0.80），同时在TR-FRET系统中保持可接受的功能（Z'=0.53）。相比之下，T2-硼二吡咯-FL在TR-FRET中提供最佳性能（Z'=0.57），但在NanoBRET中也显示出应用潜力（Z'高达0.72），尽管效率有所降低。用未标记抑制剂进行的竞争分析在所有示踪剂-平台组合中产生了一致的结合常数，验证了它们用于定量测量的可靠性。我们的研究结果突出了在不同分析平台上整合单一示踪剂的潜力，减少了单独开发探针的需求并提高了实验一致性。这种方法对于简化分析开发、提高数据可比性具有广泛意义，并能够在生化和细胞数据之间进行更直接的比较，对跨不同靶点类别的综合药物发现计划具有更广泛的意义。
