Gueret, S. M. | Search bioRxiv

bioRxiv2025

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

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 for 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 efficient 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 target engagement. O_FIG O_LINKSMALLFIG WIDTH=183 HEIGHT=200 SRC="FIGDIR/small/673904v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@191848borg.highwire.dtl.DTLVardef@94fae6org.highwire.dtl.DTLVardef@e43016org.highwire.dtl.DTLVardef@110f9f0_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

Robust prediction of relative binding energies for protein-protein complex mutations using free energy perturbation calculations

Computational free energy-based methods have the potential to significantly improve throughput and decrease costs of protein design efforts. Such methods must reach a high level of reliability, accuracy, and automation to be effectively deployed in practical industrial settings in a way that impacts protein design projects. Here, we present a benchmark study for the calculation of relative changes in protein-protein binding affinity for single point mutations across a variety of systems from the literature, using free energy perturbation (FEP+) calculations. We describe a method for robust treatment of alternate protonation states for titratable amino acids, which yields improved correlation with and reduced error compared to experimental binding free energies. Following careful analysis of the largest outlier cases in our dataset, we assess limitations of the default FEP+ protocols and introduce an automated script which identifies probable outlier cases that may require additional scrutiny and calculates an empirical correction for a subset of charge-related outliers. Through a series of three additional case study systems, we discuss how protein FEP+ can be applied to real-world protein design projects, and suggest areas of further study. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=79 SRC="FIGDIR/small/590325v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@1c5e607org.highwire.dtl.DTLVardef@1810ee5org.highwire.dtl.DTLVardef@1f8f1acorg.highwire.dtl.DTLVardef@c28053_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIReliable calculation of relative binding free energy changes for most protein mutations to within [~]1 kcal/mol. C_LIO_LIAutomated Protein FEP+ Groups treatment of alternate protonation states for titratable residues. C_LIO_LIApplication of FEP+ methodology to "real-world" protein design projects. C_LI

biophysics↗