ROTACs leverage signaling-incompetent R-spondin for targeted protein degradation

Area of Science:

• Biochemistry
• Molecular Biology
• Oncology

Background:

• Proteolysis-targeting chimeras (PROTACs) offer therapeutic potential but have limitations in targeting cell surface proteins.
• Existing PROTACs struggle to effectively degrade transmembrane proteins.

Purpose of the Study:

• Introduce ROTACs (R-spondin (RSPO) chimeras) as a novel approach to target cell surface proteins for degradation.
• Demonstrate the efficacy of ROTACs in degrading the immune checkpoint protein PD-L1.

Main Methods:

• Designed and synthesized bispecific RSPO chimeras (ROTACs) that leverage ZNRF3/RNF43 E3 ligases.
• Utilized a specific RSPO2-PD-L1 chimera (R2PD1) to target PD-L1 on melanoma cells.
• Assessed PD-L1 degradation, dependence on ZNRF3/RNF43, and anti-tumor effects in vitro.

Main Results:

• The R2PD1 chimera induced lysosomal degradation of PD-L1 at picomolar concentrations.
• Achieved 50-90% PD-L1 degradation in three melanoma cell lines, dependent on ZNRF3/RNF43.
• R2PD1 demonstrated superior inhibition of tumor cell proliferation and T-cell reactivation compared to Atezolizumab.

Conclusions:

• Signaling-disabled ROTACs provide a new paradigm for targeting cell surface proteins for degradation.
• This approach holds potential for diverse therapeutic applications, particularly in cancer immunotherapy.
• ROTACs offer a potent strategy to overcome limitations of current PROTACs for cell surface targets.