# Overcoming Ligand Discovery Challenges: Developing Peptide-Based Tracers for SPSB2

## TL;DR

This study develops peptide-based tracers for the SPSB2 E3 ligase to improve drug discovery by enabling efficient cellular delivery and target engagement assessment.

## Contribution

A novel strategy using CPP-conjugated polar degron peptides for SPSB2 ligand delivery and target engagement evaluation.

## Key findings

- Conjugating CPPs to degron peptides enabled successful cellular delivery of SPSB2 ligands.
- High-resolution crystal structure and biophysical techniques confirmed the impact of modifications on ligand behavior.
- Confocal microscopy and BRET assays validated effective target engagement in cells.

## Abstract

Developing new E3
ligase ligands for the design of heterobivalent
molecules, such as PROteolysis TArgeting Chimeras (PROTACs), requires
careful evaluation of target engagement (TE). Characterizing protein–protein
interactions (PPIs) is therefore essential in drug discovery, as it
enables the assessment of ligand binding to sites that are often difficult
to target. Degrons, peptide motifs recognized by E3 ligases, may serve
as valuable starting points for designing E3 ligands. However, many
degrons are highly polar and lack intrinsic membrane permeability,
requiring alternative strategies for efficient cellular delivery.
In this study, we used the SPRY domain-containing SOCS box protein
2 (SPSB2) E3 ligase as a model system to develop TE strategies in vitro and in cellulo using polar degron-based
peptides. By conjugating various polycationic cell-penetrating peptides
(CPPs) to the degron sequence, we present a study demonstrating cellular
delivery. We obtained a high-resolution crystal structure and used
various biophysical techniques to assess the influence of each modification,
while confocal microscopy and BRET-based assays confirmed successful
cellular delivery as well as potent TE.