Competitive, connective and allosteric Nanobodies that modulate the SOS1•RAS protein-protein interactions and tune the nucleotide exchange rate as a starting point for drug design

AbstractProtein-protein interactions (PPIs) are central in cell metabolism but research tools for the structural and functional characterization of these PPIs are often missing. Here we introduce novel and broadly applicable immunization (Cross-link PPIs andimmunizellamas, ChILL) and selection strategies (Display andco-selection, DisCO) for the discovery of diverse Nanobodies that either stabilize or disrupt PPIs in a single experiment. We applied ChILL and DisCO to identify competitive, connective or fully allosteric Nanobodies that inhibit or facilitate the formation of the SOS1•RAS complex and modulate the nucleotide exchange rate on this pivotal GTPasein vitroand RAS signallingin cellulo. One of these connective Nanobodies fills a cavity that was previously identified as the binding pocket for a series of therapeutic lead compounds. The long complementarity-determining region (CDR3) that penetrates this binding pocket serves as an innovative pharmacophore for extending the repertoire of potential leads.