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 has emerged as a novel therapeutic avenue in drug development to address and inhibit canonically difficult targets. While TPD strategies have been successful in targeting 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 five transmembrane PA-TM-RING-type E3 ubiquitin ligases (RNF128, RNF130, RNF167, RNF43, and ZNRF3), covering a broad range and selectivity of tissue expression, with which we characterized the expression in human and mouse cell lines and immune cells (PBMCs). We demonstrate that heterobifunctional REULR molecules can enforce transmembrane E3 ligase interactions with a variety of disease-relevant target receptors (EGFR, EPOR, and 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, and 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 the facile modulation of cell surface proteins by induced proximity to transmembrane PA-TM-RING E3 ligases.

Targeted protein degradation systems to enhance Wnt signaling

“…The TPD systems involving either ASGR or ZNRF3/RNF43 E3-ligases include, LYTAC containing an ASGR specific ligand fused to an antibody against a POI (protein of interest, Ahn et al, 2021); and AbTAC (Cotton et al, 2021), PROTAB (Marei et al, 2022), or REULR (Siepe et al, 2023), which are bispecific antibodies that bring E3 ligases to the proximity of a POI.…”

“…6). Therefore, from the perspective of enhanced ASGR1 degradation, the SWEETS molecules also have what are now described as AbTAC (Cotton et al, 2021), PROTAB (Marei et al, 2022), REULR (Siepe et al, 2023), and ROTAC (Sun et al, 2023) TPD activities. This E3-mediated degradation is an efficient and rapid process.…”

“…Targeted protein degraders that focus on cell surface and extracellular protein have also emerged in recent years, these include LYTAC, TransTAC, AbTAC, PROTAB, REULR, bispecific aptamer chimera, GlueTAC, and KineTAC (Zhao et al ., 2022). The TPD systems involving either ASGR or ZNRF3/RNF43 E3-ligases include, LYTAC containing an ASGR specific ligand fused to an antibody against a POI (protein of interest, Ahn et al, 2021); and AbTAC (Cotton et al, 2021), PROTAB (Marei et al, 2022), or REULR (Siepe et al, 2023), which are bispecific antibodies that bring E3 ligases to the proximity of a POI.…”

Design principles and therapeutic applications of novel synthetic WNT signaling agonists

“…Antibody-based targeting of E3 ligases and ASGR1 have been found to facilitate proteasomal degradation via ubiquitylation or lysosomal pathway degradation. 32 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 These studies have demonstrated the feasibility of cell-targeted RSPO mimetics and enabled new opportunities for the development of RSPO mimetic WNT modulators for regenerative medicine.…”

TAC‐tics for Leveraging Proximity Biology in Drug Discovery

“…A bispecific nanobody-based degrader modality was also recently reported, named REULR (receptor elimination by E3 ubiquitin ligase recruitment) (Figure 3). [28] Nanobodies are single-chain variable domain antibodies, and thus REULRs are smaller than AbTACs and PROTABs, which can offer practical and potentially pharmacokinetic advantages. Synthetic nanobodies generated against the extracellular domains of a series of RNF ligases were combined with two known EGFR nanobodies.…”