NanoB2 to monitor interactions of ligands with membrane proteins by combining nanobodies and NanoBRET

The therapeutic potential of ligands targeting disease-associated membrane proteins is predicted by ligand-receptor binding constants, which can be determined using NanoLuciferase (NanoLuc)-based biolumines-cence resonance energy transfer (NanoBRET) methods. However, the broad applicability of these methods is hampered by the restricted availability of fluorescent probes. We describe the use of antibody fragments, like nanobodies, as universal building blocks for fluorescent probes for use in NanoBRET. Our nanobody-NanoBRET (NanoB2) workflow starts with the generation of NanoLuc-tagged receptors and fluorescent nanobodies, enabling homogeneous, real-time monitoring of nanobody-receptor binding. Moreover, NanoB2 facilitates the assessment of receptor binding of unlabeled ligands in competition binding experi-ments. The broad significance is illustrated by the successful application of NanoB2 to different drug targets (e.g., multiple G protein-coupled receptors [GPCRs] and a receptor tyrosine kinase [RTK]) at distinct thera-peutically relevant binding sites (i.e., extracellular and intracellular).

Automated summary

The use of nanobodies and NanoBRET methods allows for real-time monitoring of nanobody-receptor binding, predicting the therapeutic potential of ligands targeting disease-associated membrane proteins, which is significant for drug research.