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Updated: Aug 15, 2026
A Simple Bioassay for the Evaluation of Vascular Endothelial Growth Factors
Published on: March 15, 2016
A high-sensitivity TIE2-GRB2 BRET platform for functional and pharmacologic profiling of pathogenic variants
Hitomi Matsutani1,2, Atsuro Oishi3,4, Takuya Izumi-Tamura5
1Department of Anatomy, Kyorin University School of Medicine, 181-8611, Tokyo, Japan.
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.
Insights
A new BRET biosensor effectively measures TIE2 receptor activation, aiding the development of targeted therapies for vascular malformations like venous malformations (VMs) and Blue Rubber Bleb Nevus Syndrome (BRBNS). This tool identifies potential drug candidates for these challenging conditions.
Area of Science:
- Biochemistry and Molecular Biology
- Vascular Biology
- Drug Discovery
Background:
- TIE2 receptor tyrosine kinase (RTK) is crucial for vascular integrity, with active mutants linked to venous malformations (VMs).
- Current treatments for VMs, including Blue Rubber Bleb Nevus Syndrome (BRBNS), are limited, and scalable assays for TIE2 activation are lacking.
- Existing methods like immunoblotting are not high-throughput, hindering pharmacological evaluation.
Purpose of the Study:
- To develop a novel bioluminescence resonance energy transfer (BRET) biosensor for quantifying TIE2 activation.
- To establish a high-throughput screening method for identifying TIE2-targeting drugs.
- To validate the biosensor's efficacy in identifying therapeutic candidates for VM-associated TIE2 mutants.
Main Methods:
- Developed a BRET biosensor to monitor TIE2 activation via GRB2 recruitment, a key RTK signaling adaptor.
- Utilized HEK293T cells for primary BRET screening and HUVEC-derived cells for endothelial validation.
- Assessed downstream signaling (AKT/ERK) and functional assays (tube formation) to confirm BRET findings.
Main Results:
- The BRET biosensor accurately detected angiopoietin-1 (ANG-1) and captured constitutive TIE2 mutant activity.
- Identified regorafenib and lenvatinib as potential inhibitors for the T1105N-T1106P BRBNS-associated TIE2 mutant.
- Endothelial cell assays corroborated BRET findings, confirming drug efficacy at receptor, signaling, and functional levels.
Conclusions:
- The BRET biosensor provides a scalable, two-step framework for efficient primary profiling and orthogonal validation of TIE2-targeting drug candidates.
- This approach facilitates the prioritization of pharmacological agents for further preclinical and clinical development in vascular malformations.
- The developed biosensor represents a practical strategy for advancing therapeutic options for conditions driven by aberrant TIE2 signaling.
