UCSF

Drug discovery has historically been most successful by crafting small molecules that mimic metabolites or hormones in order to perturb the function of proteins. However, many of today’s most alluring drug targets are considered “undruggable” according to this prevailing paradigm, because they lack well-defined ligand-binding pockets and instead function through protein- protein or protein-DNA interactions. These challenging drug targets are currently addressed by either directly blocking their interactions with other macromolecules or engaging distant regulatory sites, known as allosteric sites, in order to alter the conformation of the protein. Since high-throughput screening, the most commonly used drug discovery technique, is not often successful at finding ligands that disrupt macromolecular interactions or that bind to allosteric sites, there has been great interest in the development of new methods that enable direct interrogation of the ligand-binding pocket of interest. This thesis begins with a review of the utility of Tethering, a site-directed ligand discovery method, for rapidly generating prototype ligands for protein-protein interfaces (PPIs) and allosteric sites. Chapter 2 describes the adaptation of Tethering to a competitive binding assay format to enable the direct discovery of PPI antagonists. Chapter 3 describes the development of a site-directed high-throughput screen targeting an allosteric peptide-binding site on the protein kinase PDK1. The chemical optimization of these compounds is reported, along with the determination of co-crystal structures to establish their binding modes, and their capacity to inhibit PDK1 signal transduction in cells. Finally, Chapter 4, describes a general computational approach to discover small molecules that target the same PPI in PDK1 via virtual screening. Overall, this thesis provides a compelling case for using site-directed ligand discovery methods to target PPIs. In addition, our work on the protein kinase PDK1 lays the groundwork for new ligand discovery efforts targeting the same allosteric site that is present in numerous other protein kinases.