A high-sensitivity TIE2–GRB2 BRET platform for functional and pharmacologic profiling of pathogenic variants associated with venous malformations

Abstract

TIE2 is an endothelial receptor tyrosine kinase (RTK) essential for vascular integrity, and constitutively active TIE2 mutants are involved in venous malformations (VMs). VMs are currently treated by surgery or sclerotherapy, but effective pharmacologic options remain limited, especially for surgically challenging Blue Rubber Bleb Nevus Syndrome (BRBNS). TIE2 activation has typically been assessed by immunoblotting of phosphoproteins, yet scalable assays applicable to pharmacological evaluation are still lacking. Here, we present a bioluminescence resonance energy transfer (BRET) biosensor that quantifies receptor-proximal TIE2 activation by monitoring recruitment of GRB2, a major adaptor in RTK signaling. This BRET sensor detects angiopoietin-1 (ANG-1) at physiological plasma concentrations and is compatible with a high-throughput format. Furthermore, it captures constitutive activity of TIE2 mutants associated with VMs and enables pharmacological evaluation of TIE2 variants. Notably, the T1105N–T1106P variant, frequently observed in BRBNS, exhibited relatively higher sensitivity to the clinical oncology drugs regorafenib and lenvatinib. To validate selected BRET-defined pharmacological profiles in an endothelial context, we used HUVEC-derived HUEhT-2 cells and established endothelial TIE2–GRB2 BRET measurements, followed by downstream AKT/ERK signaling analysis and tube formation assays. These endothelial assays largely supported the selected HEK293T BRET findings, particularly the pharmacological profile of T1105N–T1106P mutant, at receptor-proximal, downstream signaling, and functional levels. Together, this BRET-based biosensor establishes a two-step framework: HEK293T BRET enables efficient primary profiling, whereas endothelial assays provide orthogonal validation to support biological interpretation. This framework offers a practical strategy for prioritizing pharmacological candidates for further endothelial and in vivo validation toward therapeutic development.