Cossar, P. J. | Search bioRxiv

bioRxiv2022

Early identification of cooperative fragments for protein-protein interaction stabilization

Modulating protein-protein interactions (PPIs) is an effective approach to drug discovery, with several drugs in the clinic that inhibit PPIs. The orthogonal approach of PPI stabilization has developed slowly, a function of the complicated dynamics of multi-component protein complexes. In contrast to PPI inhibition, where ligand affinity is the driving parameter for efficacy, cooperativity is frequently the directing variable for PPI stabilization. Here we show how STD NMR allows for early-stage detection of cooperativity using the hub protein 14-3-3, a focused library of fragments and several 14-3-3 partner proteins. Further, we validate that the observed enhancement in STD signal is a function of cooperativity of the ternary 14-3-3 complex, using mutagenesis and X-ray crystallography. Additionally, we assess the differential cooperativity of three fragments in a panel of 14-3-3 interaction partners. Finally, we demonstrate how selective 14-3-3 complex formation is a function of cooperativity effects

biochemistry↗

bioRxiv2021

Covalent Flexible Peptide Docking in Rosetta

Electrophilic peptides that form an irreversible covalent bond with their target have great potential for binding targets that have been previously considered undruggable. However, the discovery of such peptides remains a challenge. Here, we present CovPepDock, a computational pipeline for peptide docking that incorporates covalent binding between the peptide and a receptor cysteine. We applied CovPepDock retrospectively to a dataset of 115 disulfide-bound peptides and a dataset of 54 electrophilic peptides, for which it produced a top-five scoring, near-native model, in 89% and 100% of the cases, respectively. In addition, we developed a protocol for designing electrophilic peptide binders based on known non-covalent binders or protein-protein interfaces. We identified 7,154 peptide candidates in the PDB for application of this protocol. As a proof-of-concept we validated the protocol on the non-covalent complex of 14-3-3{sigma} and YAP1 phosphopeptide. The protocol identified seven highly potent and selective irreversible peptide binders. The predicted binding mode of one of the peptides was validated using X-ray crystallography. This case-study demonstrates the utility and impact of CovPepDock. It suggests that many new electrophilic peptide binders can be rapidly discovered, with significant potential as therapeutic molecules and chemical probes.

biochemistry↗