GoKawiilSupplementary Information file contains Supplementary Figs. 1-69, Supplementary Tables 1-11 and Supplementary References. Supplementary Figs. 1-6. Scaffold diversity of metaproteome-derived miniproteins scaffolds, design principles and computational analysis of MetaGen and RFdiffusion designs. Supplementary Figs. 7-9. OPS-RD assay development and optimization. Supplementary Figs. 10-15. Discovery, optimization, and characterization of MRGPRX1 binders. Supplementary Figs. 16-19. NK1R binder screening and pharmacological characterization. Supplementary Figs. 20-21. Cryo-EM structures of MRGPRX1-miniprotein complexes. Supplementary Figs. 22-25. CXCR4 binder screening and characterization. Supplementary Figs. 26-30. CCR5 binder screening and pharmacological characterization. Supplementary Figs. 31-32. OXTR binder identification and pharmacological characterization. Supplementary Figs. 33-39. Structural and functional characterization of CXCR4 maxibinder dCX1_001. Supplementary Figs. 40-47. Yeast display, biophysical and pharmacological characterization of GLP1R, GIPR, and GCGR binders. Supplementary Figs. 48-49. OPS-RD-based PTH1R binder identification and functional screening. Supplementary Figs. 50-60. CGRPR miniprotein in vitro and in vivo characterization. Supplementary Figs. 61-65. Structural analysis of CGRPR-miniprotein complexes and receptor mutagenesis. Supplementary Figs. 66-69. Computational analysis of design methods, in vivo studies of dCX1_001 and surface analysis of chemokine receptors. Supplementary Table 1. OPS-RD assay performance parameters across GFP-tagged GPCRs. Supplementary Tables 2-3. Yeast display and OPS-RD binding data of PAC1R binders. Supplementary Tabes 4-5. Pharmacological data of MRGPRX1 and NK1R binders. Supplementary Table 6. Contact list between MRGPRX1 and adducts at distance of < 5 Å. Supplementary Tables 7-8. Pharmacological data of CXCR4, CCR5, OXTR, GIPR and PTH1R binders. Supplementary Table 9. Binding data of PTH1R binders. Supplementary Tables 10-11. Pharmacological data of CGRPR binders and sequences of functional GPCR binders extensively characterized in pharmacological assays.
De novo design of miniproteins targeting GPCRs
Why This Matters
This study demonstrates the de novo design of miniproteins targeting GPCRs, a significant breakthrough given the challenge of developing small, stable, and specific receptor binders. These engineered miniproteins have potential applications in drug discovery, diagnostics, and therapeutics, offering new avenues for targeting complex membrane proteins with high precision. The integration of computational design, structural analysis, and functional validation marks a major advancement in protein engineering and receptor targeting technologies.
Key Takeaways
- De novo miniproteins can specifically target GPCRs, expanding therapeutic possibilities.
- Combining computational design with structural analysis accelerates receptor binder development.
- This approach opens new avenues for drug discovery and precision medicine targeting membrane proteins.
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