One Tracer, Dual Platforms: Unlocking Versatility of Fluorescent Probes in TR-FRET and NanoBRET Target Engagement Assays.

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.