A Functional Assay for Mining Noninhibitory Enzyme Ligands from One Bead One Compound Libraries: Application to E3 Ubiquitin Ligases

Chemical dimerizers are synthetic molecules that bring into proximity two or more proteins that do not normally interact with one another. A major application of this technology is to recruit an enzyme to a target protein, resulting in its post-translational modification (PTM). In particular, chemical dimerizer-mediated polyubiquitylation of proteins has garnered an enormous amount of interest as a new drug modality. A fundamental requirement for the construction of new PTM-driving dimerizers is an enzyme ligand that does not inhibit its activity. Traditional activity-based high-throughput screening platforms are not suited for this purpose. Here we describe a novel platform for screening libraries of bead-displayed compounds that links a requirement for small-molecule binding to the enzyme with enzyme-mediated modification of a nearby substrate. This system ensures that the enzyme-recruiting small molecules do not interfere with the catalytic function of the enzyme. We demonstrate the utility of this system in the context of E3 ubiquitin ligase-recruiting molecules and report the discovery of a novel low-molecular-mass ligand for the von Hippel-Lindau (VHL) protein.