Probing structural adaptivity at PPI interfaces with small molecules

There is strong interest in developing small molecules that modulate protein-protein interactions (PPI), since such compounds could serve as drug leads or as probes of protein function. Fragment-based ligand discovery has been a particularly useful approach for modulating PPI. Fragments are typically <250 Da compounds that bind to proteins with weak affinity but high ligand efficiency. Here, we review a method for fragment-based ligand discovery using covalent disulfide trapping (Tethering). Tethering uses a native or engineered cysteine residue to select thiol-containing fragments that bind to the protein near the tethering cysteine. Taking advantage of the site-directed nature of Tethering, one can investigate the ‘druggability’ of particular binding sites on a protein surface; furthermore, Tethering has been used to find new binding sites and to stabilize allosteric conformations. We review the principles of Tethering and discuss two examples where disulfide trapping has expanded our understanding of PPI. For the cytokine interleukin-2 (IL2), Tethering identified a binding site adjacent to the IL2/IL2-receptor and a new site allosterically coupled to this PPI. For the kinase PDK1, Tethering identified ligands that activated or inhibited enzymatic activity by binding to a single allosteric site. These examples provide a context for successful fragment-discovery projects, in which complementary technologies work together to identify starting points for chemical biology and drug discovery.