A combined in silico approach to design peptide ligands with increased receptor-subtype selectivity.

G-protein coupled receptors are major drug targets that change their conformation upon binding of ligands to their extracellular binding pocket to transduce the signal to intracellular G-proteins or arrestins. In drug screening campaigns, computational methods are frequently used to predict binding affinities for chemical compounds in silico before experimental testing. Some of these methods take into consideration the inherent flexibility of the ligand and to some extent also of the receptor. Due to high structural flexibility, peptide ligands are exceptionally difficult to handle and approaches to effectively sample in silico receptor-peptide ligand interactions are limited. Here we describe a pipeline starting from microseconds molecular dynamics simulations of receptor and receptor ligand complexes to find reasonable starting conformations and derive constraints for subsequent flexible docking of peptide ligands, using Rosetta's FlexPepDock approach. We applied this approach to predict binding affinities for dynorphin and its variants to members of the opioid receptor family. Using an ensemble of docking poses, Rosetta's fixbb protein design method explored the sequence space at defined positions, to enhance binding affinities, aiming to increase subtype selectivity towards κ-opioid receptor while decreasing it towards μ-opioid receptor. The results of our computations were validated experimentally in a related study (Zangrandi et al., 20241). Four out of six proposed variants lead to a significant increase in subtype selectivity in favor of κ-opioid receptor, highlighting the potential of our approach to design subtype selective peptide variants. The established workflow may also apply for other receptor types activated by peptide ligands.