Targeting intracellular PPI with membrane permeable peptides

16 AA

peptide optimized

PAMPA logPerm achieved

IC50 of the best peptide

The target is Vps29 that plays a key role in endosomal recycling through intracellular PPI.
Starting peptide of 16 AAs had low experimental permeability  (-8 logPerm) and affinity (780 nM)
Starting binding mode derived from the co crystal structure, stabilized by 6 hydrogen bonds with Glu, Val, and Tyr residues.
The ligand also forms 11 hydrophobic interactions with Lys, Arg, Leu, and Tyr.
The goal is to achieve membrane permeability and to improve affinity.

We defined the list of allowed AA substitutions per position, resulting in the design space of >1B peptide sequences.
100k compounds were screened with QuorumMap.
20 top-ranked peptides from each strategy were selected for experimental testing.

9 hits improved both affinity and permeability:
- affinity improved from 780 nM to ~250 nM;
- permeability improved from −8 to −5.92 (logPerm).
A new π-π interaction is formed in the optimized binding mode between Tyr and Pro, adding aromatic stabilization in cost of few weak hydrophobic interactions.
The number of hydrogen bonds in optimized binding mode increased to 8: with Glu, Arg, and Tyr.

Optimized binding modes

The starting peptide showed identical in-membrane and in-water conformations.
The optimized peptide showed clear chameleonicity, adapting its conformation to both membrane and water environments.
In the membrane, hydrophobic residues are exposed to the hydrophobic environment, while hydrophilic residues form a dense network of hydrogen bonds that bury polarity inside the peptide.
In water, hydrophobic regions become partially shielded, while polar residues form direct interactions with the solvent, reducing the internal hydrogen-bond network.

Allowed N-shielding positions were defined as sites where modification would not disrupt the binding geometry.
N-shielding included methyl, ethyl, propyl, and cyclopropyl groups..
The best peptide has ethyl shielding at positions 1, 7, and 9, reaching logPerm of -5.63.10

Guided N-shielding positions for improved peptide permeability.