Incorporating a β-hairpin sequence motif to increase intracellular stability of a peptide-based PROTAC.

Proteolysis targeting chimeras (PROTACs) have emerged as a new class of therapeutics that utilize the ubiquitin-proteasome system (UPS) to facilitate proteasomal degradation of "undruggable" targets. Peptide-based PROTACs contain three essential components: a binding motif for the target protein, a short amino acid sequence recognized by an E3 ligase called a degron, and a cell penetrating peptide to facilitate uptake into intact cells. While peptide-based PROTACs have been shown to successfully degrade numerous targets, they have often been found to exhibit low cell permeability and high protease susceptibility. Prior work identified peptides containing a β-hairpin sequence motif that function not only as protecting elements, but also as CPPs and degrons. The goal of this study was to investigate if a β-hairpin sequence could replace commonly used unstructured peptides sequences as the degron and the CPP needed for PROTAC uptake and function. The degradation of the protein Tau was selected as a model system as several published works have identified a Tau binding element that could easily be conjugated to the β-hairpin sequence. A series of time- and concentration-dependent studies confirmed that the βhairpin sequence was an adequate alternative CPP and degron to facilitate the proteasomemediated degradation of Tau. Microscopy studies confirmed the time-dependent uptake of the PROTAC and a degradation assay confirmed that the β-hairpin conjugated PROTAC had a greater lifetime in cells.