Abstract
Purpose :
Current treatments for retinal vascular disorders have focused on agents that inhibit vascular endothelial growth factor (VEGF), which is elevated in such conditions and plays a significant role in angiogenesis and vascular permeability. Unfortunately, not all patients respond to anti-VEGF drugs, partly due to the involvement of other proteins in angiogenesis, such as angiopoietin-1 receptor (TIE2), a tyrosine kinase that promotes vascular stability. The natural TIE2 agonist, angiopoietin-1 (ANGPT1) made by pericytes, are often damaged in retinal vascular conditions, including diabetic retinopathy. Consequently, insufficient ANGPT1 is being released to stimulate TIE2 and stabilize blood vessels. Therefore, the discovery of agents that stimulate TIE2 may be useful.
Methods :
We screened a phage display library containing clones expressing approximately one billion unique cyclic peptides. We performed three successive biopanning experiments looking for binding to recombinant human TIE2 Fc and control IgG proteins. We then used Sanger Sequencing to determine the peptides’ sequences expressed by the phage clones. An enzyme-linked immunosorbent assay confirmed high affinity binding peptides to targets. In vitro assays on Human Umbilical Vein Endothelial Cells (HUVEC) were employed to study the binding of peptides to TIE2 and subsequent TIE2 pathway stimulation.
Results :
We identified a new 12-mer cyclic peptide, TC1, which demonstrates a high affinity to TIE2. Preliminary data showed that treating HUVEC with TC1 activated TIE2 phosphorylation and also the phosphorylation of AKT, a major angiogenic mediator downstream of the TIE2 pathway. When TC1 and recombinant ANGPT1 are added together to cells, TC1 prevents ANGPT1 from binding to TIE2, showing that TC1 and ANGPT1 compete for the receptor TIE2.
Conclusions :
It is known that oligomerization and clustering are important for ANGPT1 activation of TIE2. The fact that TC1, a monomer, stimulated the TIE2 pathway could provide more insights into ANGPT1-TIE2 biology and may offer a potential therapeutic benefit to people with retinal vascular disorders. We are testing TC1 octomers using polyethylene glycol linkers to determine if enhanced clustering will increase activity.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.