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

Target engagement assays are essential for drug discovery, utilizing Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) and Nano Bioluminescence Resonance Energy Transfer (NanoBRET) as complementary methods for biochemical and cellular evaluation. Traditional platforms require distinct fluorescent tracers, increasing costs and complexity. This study systematically evaluates the cross-platform performance of T2-BODIPY-FL and T2-BODIPY-589, tracers developed for receptor-interacting protein kinase 1 (RIPK1) target engagement in TR-FRET and NanoBRET applications, respectively. Our results demonstrate both tracers effectively bridge biochemical and cellular assays, providing reliable measurements. T2-BODIPY-589 demonstrates superior performance in NanoBRET (Z' up to 0.80) and acceptable functionality in TR-FRET (Z'=0.53). Conversely, T2-BODIPY-FL performs optimally in TR-FRET (Z'=0.57) and exhibits NanoBRET potential (Z' up to 0.72). Competition assays with an unlabeled inhibitor yielded consistent binding constants across all combinations. These findings suggest a single tracer can integrate diverse assay platforms, enhancing consistency and comparability in drug discovery.