Biologics-based degraders - an expanding toolkit for targeted-protein degradation

Abstract

Targeted protein degradation (TPD) is a broadly useful proteome editing tool for biological research and therapeutic development. TPD offers several advantages over functional inhibition alone, including the ability to target previously undruggable proteins and the substantial and sustained knockout of protein activity. A variety of small molecule approaches hijack endogenous protein degradation machinery, but are limited to proteins with a cytosolic domain and suitable binding pocket. Recently, biologics-based methods have expanded the TPD toolbox by allowing access to extracellular and surface-exposed proteins and increasing target specificity. Here, we summarize recent advances in the use of biologics to deplete proteins through either the ubiquitin-proteasome system or the lysosomal degradation pathway, and discuss routes to their effective delivery as potential therapeutic interventions.

Figure 1

Biologics-based degraders utilizing the UPS for TPD. (a) Overview of bioPROTAC design. (b) Mechanism of action of bioPROTACs. Once bioPROTACs have entered the cell (either by protein or genetic delivery), they engage with both the POI and an E3 ligase, which leads to subsequent polyubiquitination of the POI and degradation by the UPS. (c) Similar to PROTACs, peptides can be engineered to engage with both the POI and an E3 ligase, leading to degradation of the POI by the UPS. (d) Antibody-fused PROTACs consist of an antibody targeting a cell surface receptor that is chemically linked to a small molecule PROTAC targeting an intracellular POI. After internalization, the intermolecular linkage is hydrolyzed, allowing the PROTAC to escape the lysosomal degradation pathway. The PROTAC then induces polyubiquitination to orchestrate degradation of the POI.

Figure 2

Biologics-based degraders utilizing the lysosomal degradation pathway for TPD. (a) Sweeping antibodies sequester circulating antigens and direct them toward intracellular destruction. The antibodies themselves avoid degradation by binding to FcRn, whereas an engineered pH-sensitive interaction of their variable regions enables release of the target antigen within maturing endosomes. (b) Seldegs can remove unwanted antibodies for medical or screening purposes and are degraded along with the target antigen in the lysosome. (c) LYTACs divert extracellular or transmembrane proteins toward the lysosomal degradation pathway using glycopeptides that interact with surface LTRs. Several alternative POI-binding modules have been successfully reported, including biologics, small molecules, and peptides. (d) GlueTACs are binding modules that cross-link to transmembrane POIs using a nonnatural amino acid. Internalization and lysosomal sorting are then driven by small peptides fused to the GlueTAC. Unlike the other approaches shown here, no receptor is required for internalization. (e) AbTACs co-recruit a surface-exposed POI and a transmembrane E3 ligase. The POI is ubiquitinated and directed down the lysosomal degradation pathway. Intriguingly, AbTACs appear to act in a catalytic manner, since POI degradation is not accompanied by stoichiometric loss of the E3 ligase nor the AbTAC itself.

Figure 3

Protein and genetic approaches to deliver biologics-based degraders into the cell. (a) A CPP is conjugated to the degrader to facilitate delivery across the membrane into the cell. (b) Microinjection is the direct introduction of the degrader into the cell using a micropipette. (c) Electroporation is a brief shock of electricity that induces pores in the cell membrane, allowing the degrader to enter. (d) Viral vectors or virus-like particles are used to deliver genetic material into the cell and can be used to create a stable cell line. (e) DNA is transfected into the cell to stably or transiently express the degrader. (f) mRNA is transfected into the cell to transiently express the degrader.