Receptor Elimination by E3 Ubiquitin Ligase Recruitment (REULR): A Targeted Protein Degradation Toolbox

Abstract

In recent years, targeted protein degradation (TPD) of plasma membrane proteins by hijacking the ubiquitin proteasome system (UPS) or the lysosomal pathway have emerged as novel therapeutic avenues in drug development to address and inhibit canonically difficult targets. While TPD strategies have been successful to target cell surface receptors, these approaches are limited by the availability of suitable binders to generate heterobifunctional molecules. Here, we present the development of a nanobody (VHH) based degradation toolbox termed REULR (Receptor Elimination by E3 Ubiquitin Ligase Recruitment). We generated human and mouse cross-reactive nanobodies against 5 transmembrane PA-TM-RING type E3 Ubiquitin Ligases (RNF218, RNF130, RNF167, RNF43, ZNRF3) covering a broad range and selectivity of tissue expression with which we characterized expression in human and mouse cell lines and immune cells (PBMCs). We demonstrate that heterobifunctional REULR molecules can enforce transmembrane E3 ligase interaction with a variety of disease relevant target receptors (EGFR, EPOR, PD-1) by induced proximity resulting in effective membrane clearance of the target receptor at varying levels. In addition, we designed E3 Ligase self-degrading molecules, "fratricide" REULRs (RNF128, RNF130, RENF167, RNF43, ZNRF3), that allow downregulation of one or several E3 Ligases from the cell surface and consequently modulate receptor signaling strength. REULR molecules represent a VHH-based, modular and versatile "mix and match" targeting strategy for facile modulation of cell surface proteins by induced proximity to transmembrane PA-TM-RING E3 ligases.

CYpHER: Catalytic extracellular targeted protein degradation with high potency and durable effect

“…This is mainly through formation of ternary complexes between target protein and degradation effector. For intracellular (e.g., molecular glue and PROTAC) (11) and some extracellular (e.g., AbTAC and REULR) (12, 13) molecules, this is through E3 ligase recruitment, inducing ubiquitin-mediated target degradation by the proteasome or lysosome. This benefits from a catalytic mechanism of action (the drug is not expended in the process) but can be complicated by finding an E3 ligase expressed in the target tissue that can be induced to interact in an effective orientation with the target for functional ubiquitination.…”

Directed Evolution of Genetically Encoded LYTACs for Cell-Mediated Delivery

“…The first generation of this technology came in the form of lysosome-targeting chimeras (LYTACs), which are bifunctional molecules comprised of an antibody that binds to a cell surface or secreted protein of interest (POI) conjugated to a ligand that binds a lysosome trafficking receptor such as the insulin growth factor 2 receptor (IGF2R, also known as CI-M6PR) (Figure 1A) (2,3,4). Since then, other technologies have been developed, such as AbTACs (5), ProTABs (6), and KineTACs (7), among others (8,9,10,11,12,13,14,15,16,17,18, 19, 20, 21, 22). These use similar bifunctional molecules to recruit POIs either to lysosome trafficking receptors or plasma membrane-associated ubiquitin ligases and have opened a new chapter in the field of targeted protein degradation.…”

Directed evolution of genetically encoded LYTACs for cell-mediated delivery

“…1 A ) ( 2 – 4 ). Since then, other technologies have been developed, such as AbTACs ( 5 ), ProTABs ( 6 ), and KineTACs ( 7 ), among others ( 8 – 22 ). These use similar bifunctional molecules to recruit POIs either to lysosome trafficking receptors or plasma membrane-associated ubiquitin ligases and have opened a promising direction in the field of targeted protein degradation.…”