NanoBRET-Based Biosensor for High-Throughput Screening of RAB11A–FIP2 Interaction Inhibitors

Protein-protein interactions (PPIs) are critical regulators of cellular processes and represent promising therapeutic targets. However, existing drug discovery approaches for PPI targeting, such as surface plasmon resonance, have inherent limitations, including their inability to fully recapitulate the cellular environment. To address these challenges, we developed a NanoBRET-based RAB11A activation biosensor that utilizes bioluminescence resonance energy transfer (BRET) to detect PPIs in live cells while preserving native protein function. Using this platform, we screened a library of 7040 small molecules and identified potent inhibitors that significantly disrupted RAB11A binding to its effector proteins, as indicated by a reduction in the BRET ratio. RAB11A is a key regulator of recycling endosomes, particularly in glucose transporter (GLUT) trafficking, making it an attractive target for metabolic intervention. Given that RAB11 family interacting protein 2 (FIP2) binding is essential for RAB11A activation, disrupting this interaction impairs GLUT3 trafficking and reduces glucose uptake, a critical metabolic process driving glioblastoma progression. We found that inhibition of the RAB11A-FIP2 axis attenuates GLUT3 trafficking, thereby impairing glucose metabolism in glioblastoma. Among the identified small molecules, three candidates were further validated to interfere with both RAB11A activation and GLUT3 trafficking, highlighting their potential as metabolic regulators. Our NanoBRET-based biosensor provides a robust and scalable platform for high-throughput screening of PPI inhibitors, offering a promising strategy for developing novel therapeutics targeting PPIs.