insight - Computational Biology - # Targeted Protein Degradation of Asialoglycoprotein Receptor for Wnt Signaling Modulation

Discovery and Characterization of Novel Antibodies Targeting the Asialoglycoprotein Receptor for Tissue-Specific Protein Degradation and Wnt Signaling Enhancement

Q: How could the SWEETS platform be further optimized to achieve more specific degradation of target proteins while minimizing unintended effects on other receptors like ASGR1

To optimize the SWEETS platform for more specific degradation of target proteins while minimizing unintended effects on other receptors like ASGR1, several strategies can be employed: Epitope Engineering: By designing antibodies with epitopes that are unique to the target protein and do not overlap with other receptors like ASGR1, specificity can be enhanced. This can involve structural analysis to identify regions on the target protein that are distinct from those on off-target receptors. Linker Optimization: The linker connecting the RSPO2RA domain to the antibody can be modified to ensure proper orientation and distance between the target protein and the E3 ligase. This can help in directing the degradation machinery specifically towards the target protein. Stoichiometry Control: Adjusting the stoichiometry of the SWEETS molecules, such as the ratio of RSPO2RA to the antibody, can impact the efficiency and specificity of degradation. Fine-tuning this ratio based on the target protein and receptor interactions can improve specificity. E3 Ligase Selection: Choosing E3 ligases that have high affinity for the target protein but low affinity for other receptors can help in achieving selective degradation. Screening different E3 ligases and optimizing their binding properties can enhance specificity. Validation Studies: Conducting thorough validation studies in cell lines and animal models to assess the specificity and off-target effects of the SWEETS platform. This can involve comparing the effects of SWEETS on different receptors and analyzing downstream signaling pathways to ensure specificity. By implementing these optimization strategies, the SWEETS platform can be tailored to achieve more precise and targeted degradation of specific proteins while minimizing unintended effects on other receptors.

Q: What other cell surface receptors or extracellular proteins could be targeted using a similar SWEETS-like approach, and what therapeutic applications might this enable

The SWEETS-like approach can be extended to target a variety of cell surface receptors or extracellular proteins for different therapeutic applications. Some potential targets and their therapeutic applications include: EGFR (Epidermal Growth Factor Receptor): Targeting EGFR with a SWEETS-like approach could be beneficial in cancer therapy, where EGFR overexpression is common. Enhanced degradation of EGFR could lead to inhibition of cell proliferation and tumor growth. PD-1 (Programmed Cell Death Protein 1): Modulating PD-1 levels using a SWEETS platform could enhance immune response in cancer immunotherapy. By targeting PD-1 for degradation, immune checkpoint blockade could be achieved, leading to improved anti-tumor immunity. Insulin Receptor: Targeting the insulin receptor with a SWEETS-like approach could be explored in diabetes treatment. By regulating insulin receptor levels, insulin sensitivity and glucose metabolism could be modulated, offering a novel approach to managing diabetes. VEGFR (Vascular Endothelial Growth Factor Receptor): Modulating VEGFR levels using a SWEETS platform could be beneficial in angiogenesis-related diseases. By targeting VEGFR for degradation, abnormal blood vessel formation could be controlled, offering potential therapeutic benefits in conditions like cancer and macular degeneration. By targeting different cell surface receptors or extracellular proteins with a SWEETS-like approach, a wide range of therapeutic applications in various diseases can be explored, offering new avenues for precision medicine and targeted therapies.

Q: Given the potential dual benefits of Wnt signaling enhancement and ASGR1 degradation, how could the SWEETS platform be leveraged to treat diseases with comorbidities, such as liver disease and cardiovascular disease

The dual benefits of Wnt signaling enhancement and ASGR1 degradation offered by the SWEETS platform present unique opportunities for treating diseases with comorbidities, such as liver disease and cardiovascular disease. Here are some ways the SWEETS platform could be leveraged for such conditions: Liver Disease and Cardiovascular Disease: In conditions where liver disease and cardiovascular disease coexist, the SWEETS platform could be used to target ASGR1 for degradation, potentially reducing the risk of cardiovascular events associated with ASGR1. At the same time, the enhanced Wnt signaling could promote liver regeneration and function, addressing the underlying liver disease. Diabetes and Cardiovascular Disease: By targeting specific receptors involved in glucose metabolism and cardiovascular health, the SWEETS platform could offer a dual benefit in managing diabetes and reducing the risk of cardiovascular complications. For example, targeting insulin receptors and receptors involved in vascular health could provide a comprehensive approach to managing both conditions. Cancer and Cardiovascular Disease: In cancer patients with cardiovascular comorbidities, the SWEETS platform could be used to target cancer-specific receptors for degradation while also addressing cardiovascular risk factors. This dual approach could improve cancer outcomes while mitigating cardiovascular complications. By strategically leveraging the dual benefits of Wnt signaling enhancement and targeted protein degradation offered by the SWEETS platform, innovative treatment strategies for diseases with comorbidities can be developed, potentially improving patient outcomes and quality of life.

Core Concepts

Novel antibodies targeting the asialoglycoprotein receptor (ASGR) can be used to develop a targeted protein degradation platform that enhances Wnt signaling in a tissue-specific manner.

Abstract

The content describes the discovery and characterization of two new antibodies, 8M24 and 8G8, that bind to the carbohydrate recognition domain (CRD) of the human asialoglycoprotein receptor (ASGR) subunits ASGR1 and ASGR1/2, respectively. High-resolution crystal structures of the ASGR1:8M24 and ASGR2:8G8 complexes revealed that the antibodies bind to distinct epitopes on the receptors, away from the substrate binding site.

The authors demonstrate that fusion of the mutant RSPO2 (RSPO2RA) domain to these ASGR-targeting antibodies (8M24-RSPO2RA and 8G8-RSPO2RA) results in robust Wnt signaling enhancement in cell-based assays, similar to their previously described 4F3-RSPO2RA SWEETS molecule. This is achieved through the ASGR-mediated endocytosis and degradation of the E3 ubiquitin ligases ZNRF3 and RNF43, which are negative regulators of Wnt signaling.

Interestingly, the authors also found that the SWEETS molecules can induce the degradation of ASGR1 itself, through both proteasomal and lysosomal pathways. This is likely due to the SWEETS-mediated recruitment of E3 ligases to the proximity of ASGR1, leading to its ubiquitination and subsequent degradation. The ability to degrade ASGR1 may have therapeutic implications, as reduced ASGR1 levels have been associated with a lower risk of coronary artery disease.

Overall, the study demonstrates the versatility of the SWEETS platform, which can function as a targeted protein degradation system to modulate Wnt signaling in a tissue-specific manner, while also potentially offering an approach to eliminate the ASGR1 receptor.

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biorxiv.org

Stats

"ASGR1 is estimated to be present at ~1 million copies per human hepatocyte."
"Treatment with SWEETS molecules resulted in up to ~80% reduction in ASGR1 levels within 8 hours."

Quotes

"Fusion of RSPO2RA to the anti-ASGR1 antibody 4F3 (4F3-RSPO2RA, also termed 4F3-SWEETS), which binds to the stalk region of ASGR1 outside of the CRD, leads to hepatocyte-specific RSPO mimetic activity."
"SWEETS molecules embody multiple 'TAC' activities, including LYTAC, AbTAC/PROTAB/REULR, and protein stabilization functions."

Key Insights Distilled From

Targeted protein degradation systems to enhance Wnt signaling

by Sampathkumar... at www.biorxiv.org 12-08-2023

https://www.biorxiv.org/content/10.1101/2023.12.08.570758v2

Deeper Inquiries

How could the SWEETS platform be further optimized to achieve more specific degradation of target proteins while minimizing unintended effects on other receptors like ASGR1

To optimize the SWEETS platform for more specific degradation of target proteins while minimizing unintended effects on other receptors like ASGR1, several strategies can be employed:

Epitope Engineering: By designing antibodies with epitopes that are unique to the target protein and do not overlap with other receptors like ASGR1, specificity can be enhanced. This can involve structural analysis to identify regions on the target protein that are distinct from those on off-target receptors.

Linker Optimization: The linker connecting the RSPO2RA domain to the antibody can be modified to ensure proper orientation and distance between the target protein and the E3 ligase. This can help in directing the degradation machinery specifically towards the target protein.

Stoichiometry Control: Adjusting the stoichiometry of the SWEETS molecules, such as the ratio of RSPO2RA to the antibody, can impact the efficiency and specificity of degradation. Fine-tuning this ratio based on the target protein and receptor interactions can improve specificity.

E3 Ligase Selection: Choosing E3 ligases that have high affinity for the target protein but low affinity for other receptors can help in achieving selective degradation. Screening different E3 ligases and optimizing their binding properties can enhance specificity.

Validation Studies: Conducting thorough validation studies in cell lines and animal models to assess the specificity and off-target effects of the SWEETS platform. This can involve comparing the effects of SWEETS on different receptors and analyzing downstream signaling pathways to ensure specificity.

By implementing these optimization strategies, the SWEETS platform can be tailored to achieve more precise and targeted degradation of specific proteins while minimizing unintended effects on other receptors.

What other cell surface receptors or extracellular proteins could be targeted using a similar SWEETS-like approach, and what therapeutic applications might this enable

The SWEETS-like approach can be extended to target a variety of cell surface receptors or extracellular proteins for different therapeutic applications. Some potential targets and their therapeutic applications include:

EGFR (Epidermal Growth Factor Receptor): Targeting EGFR with a SWEETS-like approach could be beneficial in cancer therapy, where EGFR overexpression is common. Enhanced degradation of EGFR could lead to inhibition of cell proliferation and tumor growth.

PD-1 (Programmed Cell Death Protein 1): Modulating PD-1 levels using a SWEETS platform could enhance immune response in cancer immunotherapy. By targeting PD-1 for degradation, immune checkpoint blockade could be achieved, leading to improved anti-tumor immunity.

Insulin Receptor: Targeting the insulin receptor with a SWEETS-like approach could be explored in diabetes treatment. By regulating insulin receptor levels, insulin sensitivity and glucose metabolism could be modulated, offering a novel approach to managing diabetes.

VEGFR (Vascular Endothelial Growth Factor Receptor): Modulating VEGFR levels using a SWEETS platform could be beneficial in angiogenesis-related diseases. By targeting VEGFR for degradation, abnormal blood vessel formation could be controlled, offering potential therapeutic benefits in conditions like cancer and macular degeneration.

By targeting different cell surface receptors or extracellular proteins with a SWEETS-like approach, a wide range of therapeutic applications in various diseases can be explored, offering new avenues for precision medicine and targeted therapies.

Given the potential dual benefits of Wnt signaling enhancement and ASGR1 degradation, how could the SWEETS platform be leveraged to treat diseases with comorbidities, such as liver disease and cardiovascular disease

The dual benefits of Wnt signaling enhancement and ASGR1 degradation offered by the SWEETS platform present unique opportunities for treating diseases with comorbidities, such as liver disease and cardiovascular disease. Here are some ways the SWEETS platform could be leveraged for such conditions:

Liver Disease and Cardiovascular Disease: In conditions where liver disease and cardiovascular disease coexist, the SWEETS platform could be used to target ASGR1 for degradation, potentially reducing the risk of cardiovascular events associated with ASGR1. At the same time, the enhanced Wnt signaling could promote liver regeneration and function, addressing the underlying liver disease.

Diabetes and Cardiovascular Disease: By targeting specific receptors involved in glucose metabolism and cardiovascular health, the SWEETS platform could offer a dual benefit in managing diabetes and reducing the risk of cardiovascular complications. For example, targeting insulin receptors and receptors involved in vascular health could provide a comprehensive approach to managing both conditions.

Cancer and Cardiovascular Disease: In cancer patients with cardiovascular comorbidities, the SWEETS platform could be used to target cancer-specific receptors for degradation while also addressing cardiovascular risk factors. This dual approach could improve cancer outcomes while mitigating cardiovascular complications.

By strategically leveraging the dual benefits of Wnt signaling enhancement and targeted protein degradation offered by the SWEETS platform, innovative treatment strategies for diseases with comorbidities can be developed, potentially improving patient outcomes and quality of life.