De novo designed bifunctional proteins for targeted protein degradation

Abstract

Targeted protein degradation (TPD) is a therapeutic strategy to remove disease-causing proteins by routing them to the ubiquitin-proteasome, autophagy, or lysosme machineries. For instance, proteolysis-targeting chimeras (PROTACs) are synthetic hetero-bifunctional small molecules that simultaneously bind the target and an E3 ubiquitin ligase to drive ubiquitination and degradation by the proteasome. Despite considerable success, designing such molecules is challenging and the number of currently addressable ubiquitin E3 ligases is limited. Here we demonstrate hetero-bifunctional de novo designed proteins as alternatives for TPD to access more targets and ligases. First, we develop a stable and highly adaptable helix-turn-helix scaffold for presenting different binding sites. Next, we use computational protein design to incorporate and embellish hot-spot-binding sites to target BCL-xL, plus short linear motifs (SLiMs) for KLHL20 ligase recruitment. The resulting mono- and bi-functionalised proteins bind the targets in vitro, and the latter degrade BCL-xL in cells leading to apoptosis.