A Functional Assay for Mining Noninhibitory Enzyme Ligands from One Bead One Compound Libraries: Application to E3 Ubiquitin Ligases
Weijun Gui, Allison Goss, Thomas Kodadek
Abstract
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.