Inducing Receptor Degradation as a Novel Approach to Target CC Chemokine Receptor 2 (CCR2)
Abstract
:1. Introduction
2. Results
2.1. Insertion of HaloTag7 to CCR2 Alters Receptor Function but Not Ligand Binding
2.2. HaloPROTAC3 Induces Degradation of CCR2-HaloTag-HiBiT
2.3. Real-Time HaloPROTAC3-Induced Degradation of CCR2
2.4. HaloPROTAC3 Induces Proteasomal Degradation of CCR2-HaloTag-HiBiT
2.5. Immunofluorescence Studies Confirm CCR2-HaloTag-HiBiT Degradation by HaloPROTAC3
2.6. Degradation of CCR2-HaloTag-HiBiT Translates to Reduced Receptor Activation
3. Discussion
4. Materials and Methods
4.1. Materials
4.2. Cell Culture
4.3. Transfection and Lentivirus Generation
4.4. Transduction and Clonal Isolation
4.5. Membrane Preparation
4.6. Radioligand Binding Assays
4.7. Label-Free Phenotypic Whole-Cell Assay (xCELLigence)
4.8. Lytic HiBiT Detection
4.9. Real-Time, Live Cell HiBiT Detection
4.10. Immunofluorescence
4.11. Data Analysis and Statistics
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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pIC50 ± SEM (IC50, nM) 1 | |||
---|---|---|---|
Compound | CCR2 2 | CCR2-HiBiT 3 | CCR2-HaloTag-HiBiT 3 |
CCR2-RA-[R] | 7.9 ± 0.03 (13) | 7.8 ± 0.06 (17) | 7.9 ± 0.08 (13) |
INCB3344 | 8.2 ± 0.13 (6) | 8.2 ± 0.13 (7) | 8.1 ± 0.10 (8) |
SD-24 | 8.0 ± 0.06 (10) | 8.3 ± 0.05 (5) | 8.2 ± 0.05 (6) |
Compound | pDC50/pEC50 ± SEM (DC50/EC50, nM) 1,2 | Dmax ± SEM (%) 1,3 |
---|---|---|
HaloPROTAC3 | 7.4 ± 0.01 (42) | 38 ± 5 |
ent-HaloPROTAC3 | n.a. 4 | −1 ± 4 |
INCB3344 | 8.4 ± 0.3 (6) | −20 ± 5 |
CCR2-RA-[R] | 7.8 ± 0.1 (15) | −49 ± 5 |
SD-24 | 6.8 ± 0.1 (181) | −48 ± 6 |
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Ortiz Zacarías, N.V.; Röth, S.; Broekhuis, J.D.; van der Es, D.; Moreau, K.; Heitman, L.H. Inducing Receptor Degradation as a Novel Approach to Target CC Chemokine Receptor 2 (CCR2). Int. J. Mol. Sci. 2024, 25, 8984. https://doi.org/10.3390/ijms25168984
Ortiz Zacarías NV, Röth S, Broekhuis JD, van der Es D, Moreau K, Heitman LH. Inducing Receptor Degradation as a Novel Approach to Target CC Chemokine Receptor 2 (CCR2). International Journal of Molecular Sciences. 2024; 25(16):8984. https://doi.org/10.3390/ijms25168984
Chicago/Turabian StyleOrtiz Zacarías, Natalia V., Sascha Röth, Jeremy D. Broekhuis, Daan van der Es, Kevin Moreau, and Laura H. Heitman. 2024. "Inducing Receptor Degradation as a Novel Approach to Target CC Chemokine Receptor 2 (CCR2)" International Journal of Molecular Sciences 25, no. 16: 8984. https://doi.org/10.3390/ijms25168984