Design of High-Affinity Stapled Peptides To Target the Repressor Activator Protein 1 (RAP1)/Telomeric Repeat-Binding Factor 2 (TRF2) Protein–Protein Interaction in the Shelterin Complex

Shelterin,
a six-protein complex, plays a fundamental role in protecting
both the length and the stability of telomeres. Repressor activator
protein 1 (RAP1) and telomeric repeat-binding factor 2 (TRF2) are
two subunits in shelterin that interact with each other. Small-molecule
inhibitors that block the RAP1/TRF2 protein–protein interaction
can disrupt the structure of shelterin and may be employed as pharmacological
tools to investigate the biology of shelterin. On the basis of the
cocrystal structure of RAP1/TRF2 complex, we have developed first-in-class
triazole-stapled peptides that block the protein–protein interaction
between RAP1 and TRF2. Our most potent stapled peptide binds to RAP1
protein with a Ki value of 7 nM and is
>100 times more potent than the corresponding wild-type TRF2 peptide.
On the basis of our high-affinity peptides, we have developed and
optimized a competitive, fluorescence polarization (FP) assay for
accurate and rapid determination of the binding affinities of our
designed compounds and this assay may also assist in the discovery
of non-peptide, small-molecule inhibitors capable of blocking the
RAP1/TRF2 protein–protein interaction.