Abstract
G-protein-coupled receptors (GPCRs) have key roles in physiology and are central targets for drug discovery and development1,2, but the design of protein agonists and antagonists has been challenging as GPCRs are integral membrane proteins and conformationally dynamic3,4,5,6. Here we describe de novo design methods and a high-throughput receptor-diversion microscopy-based screen for generating GPCR-binding miniproteins with high affinity, potency and selectivity. We design miniprotein agonists that activate receptors involved in itch and pain, as well as antagonists that inhibit receptors implicated in cancer, metabolic disorders such as diabetes and obesity, and migraines. The cryo-electron microscopy (cryo-EM) structures of five receptor-bound designs are close to the computational design models. A designed chemokine receptor antagonist mobilizes haematopoietic stem and progenitor cells in vivo at a level comparable to a clinically used drug, with fewer adverse effects.
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Data availability
Cryo-EM maps and models have been deposited in the Electron Microscopy Data Bank (EMDB) and PDB under the following accession codes: mM1_068/MRGPRX1: EMD-70205 and 9O7N; mM1_060/MRGPRX1: EMD-70230 and 9O8L; dC2_049/GCRPR: EMD-48385 and 9MM5; dC2_050/CGRPR: EMD-48424 and 9MNI; dCX1_001/CXCR4: EMD-68747 and 22XC. MetaGen-derived scaffolds are available online (https://files.ipd.uw.edu/pub/metagen_scaffolds/8920_scaffolds.tar.gz). OPS-RD data are available online (https://files.ipd.uw.edu/pub/GPCRs/OPS_RD/MRGPRX1.zip, https://files.ipd.uw.edu/pub/GPCRs/OPS_RD/PAC1R.zip and https://files.ipd.uw.edu/pub/GPCRs/OPS_RD/PTH1R.zip). The NGS data have been deposited in the NCBI database under accession code PRJNA1451936 with raw sequencing reads available in the Sequence Read Archive. Source data are provided with this publication.
Code availability
The Rosetta macromolecular modelling suite (https://www.rosettacommons.org) is freely available to academic and non-commercial users. Commercial licences for the suite are available through the University of Washington Technology Transfer Office. Code for using RFdiffusion v.1.0 is available at GitHub (https://github.com/RosettaCommons/RFdiffusion#binder-design). Scripts for ProteinMPNN, ProteinMPNN-FastRelax and AF2 with initial guess are available at GitHub (https://github.com/dauparas/ProteinMPNN and https://github.com/nrbennet/dl_binder_design, respectively). Code for running partial and iterative partial RFdiffusion v.1.0 is available at GitHub (https://github.com/RosettaCommons/RFdiffusion#partial-diffusion and https://github.com/davidekim/ppi_iterative_opt, respectively). Code used for estimating yeast SC50 values from FACS and NGS data can be found online (https://files.ipd.uw.edu/pub//estimate_affinity/estimate_affinity_from_ngs_sc50er.py). MotionCor3 for motion correction and the EPU_group_AFIS.py script are available at GitHub (https://github.com/czimaginginstitute/MotionCor3 and https://github.com/DustinMorado/EPU_group_AFIS, respectively).
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Acknowledgements
We thank L. Goldschmidt and K. VanWormer, respectively, for maintaining computational and wet laboratory resources at the Institute for Protein Design; B. Trippe for assistance with the analysis of yeast display data; N. Banerjee and D. Tiwari for expressing and purifying CXCR4; G. Nair for assistance with pharmacological assays; U. Wahlers for supporting protein production; M. Mohr for supporting operations at Skape Bio; I. Cheeseman and P. I. Imoukhuede for providing HeLa and HEK293/OXTR cells, respectively; A. Rudebeck for supporting computational resources at the BioInnovation Institute; M. M. Rosenkilde for discussions concerning pharmacology; A. Tongson, J. Walters, G. Leung, Q. Wang and P. Gonzales for supporting pharmacological characterization of MRGPRX1 designs; E. Rivera de Torre for assisting in CD studies; R. Christensen for technical in vivo support at Lundbeck; and our colleagues at the NNRCC. A.K.S. is a recipient of the Sonu Agrawal Memorial Chair from IIT Kanpur.
Funding
E.M. discloses support for the research of this work from Austrian Science Fund (FWF), Erwin Schrödinger program (J-4663); C.N., D.F., A.M.-B., P.R.-S., F.D. and E.P.T.H. from BioInnovation Institute Foundation (BII22SG1021010, BII24SG1021475, BII24SG1022030); C.N. from Novo Nordisk Foundation (NNF18OC0030446); D.B. and G.R.L. from the Howard Hughes Medical Institute (GR020267); E.M. and D.B. from Novo Nordisk (GR018355); D.B. from the Defense Threat Reduction Agency (HDTRA1-21-1-0038), Microsoft (GF117374, Microsoft Protein Prediction Research), The Audacious Project at the Institute for Protein Design (PG117878, PG117879, PG117866), The Nordstrom Barrier Institute for Protein Design Directors Fund (GF124659) and The Open Philanthropy Project Universal Flu Vaccine Fund (GF129461); D.B., S.V.T. and G.R.L. from The Open Philanthropy Project Improving Protein Design Fund (GF129460); D.B. and T.S. from The Wu Tsai Protein Innovation Fund (GF151772); A.M. from Cancer Research Grand Challenge provided by Cancer Research UK (GR050755); D.B. from Department of the Defense and Defense Threat Reduction Agency (HDTRA1-21-1-0007, GR013444); D.B. and J.Z.Z. from the National Institutes of Health’s National Cancer Institute (R01CA240339, GR009231, K99-CA293001) and the National Institutes of Health’s National Institute on Aging (R01AG063845, GR009173); D.B. from the US Department of Energy, Office of Science, including resources of the National Energy Research Scientific Computing Center (BER-ERCAP0022018); B.E.K. and B.L.R. from National Institutes of Health NIDA (R01DA055656) and the NIMH Psychoactive Drug Screening Program; P.M.S. and D.W. from National Health and Medical Research Council of Australia Investigator (2025694, 2026300); J.B.S. from NSF (2143160), Department of Defense (W81XWH-21-1-0891), NIH NIDCR (R21DE031436) and CureSearch for Children’s Cancer award. The research on chemokine receptors and de novo protein design in the A.K.S. laboratory is supported by a senior fellowship of the DBT Wellcome Trust India Alliance (IA/S/20/1/504916), ANRF (ANRF/ARG/2025/007393/LS), Department of Biotechnology (BT/PR53019/MED/30/2528/20) and the Lady Tata Memorial Trust. The cryo-EM structure of dCX1_001-CXCR4 was determined at the ANRF-Sponsored National cryo-EM Facility at IIT Kanpur supported by ANRF/SERB (IPA/2020/000405). The work in C.G.T.’s laboratory was supported by core funding from the Medical Research Council (MRC U105197215).
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E.M., D.F., K.D., C.G.T., C.N. and D.B. conceptualized the study. D.E.K., T.S., M.S.B., I.S. and X.W. developed computational design pipelines using RFdiffusion. C.N. and D.F. developed computational design pipelines using MetaGen and partial RFdiffusion. E.M., D.F., D.E.K., X.Q., A.P., J.E.S., C.N. and M.J. designed binders. A.N. and M.J. performed meta-analysis of designed binders. D.F. and C.N. conceived the OPS-RD HTS assay. D.F., A.M.-B., P.R.-S. and E.P.T.H. developed cell lines and performed the OPS-RD assay. E.M., X.Q., A.M.-B., D.E.K., T.S., L.M., S.V.T., P.P., Y.R. and C.N. expressed and purified binders. E.M., X.Q., F.D., P.K., P.N.H.T., L.O., W.C., N.L., M.B., Y.W., L.A., J.H.V., S.M., K.S., A.D. and S.S. pharmacologically characterized binders. J.F. and L.H. performed biofloating assay. E.M., X.Q., J.Z.Z., A.M., L.T., G.R.L., I.G., D.K.V., L.D. and A.B. performed yeast display experiments. B.C. improved the yeast SC50 estimation method from FACS and NGS. J.C., B.P.C., M.J.B. and P.N.H.T. determined the cryo-EM structure of CGRPR binders, performed the CGRPR mutagenesis and generated associated figures. Q.C. performed structural characterization of dCX1_001, with and without a rigid fusion, bound to monomeric or dimeric CXCR4. R.B. and M.G. determined the cryo-EM structure of dCX1_001 bound to homotrimeric CXCR4. B.L.R. and B.E.K. determined cryo-EM structure of MRGPRX1 binders. J.S., T.D. and X.Y. expressed and purified PAC1R and NK1R. E.P.T.H., L.J.S., J.J.W., J.F.B., C.B. and A.A.A. designed constructs with improved PK properties and conducted in vivo studies. K.V.K. performed in vivo studies with dCX1_001 antagonist of CXCR4. E.M. and C.N. drafted the work and prepared figures. E.M., D.F., D.E.K., X.Q., A.M.-B., P.R.-S., J.H.V., E.P.T.H., M.J., B.P.C., P.K., Q.C., J.F., S.M., A.D., S.S., K.V.K., N.L., M.B., Y.W., K.D., L.D., A.B., J.G.E., J.B.S., A.L., A.K.S., P.M.S., B.L.R., B.E.K., D.W., C.G.T., C.N. and D.B. reviewed, edited and revised the work. E.M., P.H., K.D., J.G.E., J.B.S., A.L., A.K.S., P.M.S., B.E.K., B.L.R., D.W., C.G.T., C.N. and D.B. provided research funding and supervision.
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E.M., D.F., D.E.K., X.Q., A.M.-B., P.R.-S., J.H.V., E.P.T.H., M.J., F.D., J.F., K.D., N.L., M.B., Y.W., L.J.S., J.B.S., C.N. and D.B. are listed as inventors or major contributors on records of innovation and associated provisional patent applications relating to discoveries described in this Article, at the University of Washington and Skape Bio. The Baker laboratory has received sponsored research funding from Novo Nordisk in support of the GLP1R, PAC1R, NK1R and PTH1R research described in this Article. A.B., L.D., N.L, M.B., J.S., K.D. and Y.W. were employees of Novo Nordisk at the time of the work, and some hold minor shareholdings as part of their employment. E.M., D.F., D.E.K., A.M.-B., P.R.-S., L.J.S., C.N., E.P.T.H., M.J, J.H.V., A.N., C.G.T., B.L.R. and D.B. are shareholders of Skape Bio. Skape Bio is seeking to commercialize aspects of the research described in this Article. B.L.R. is a member of the scientific advisory board of ImprintBio, XyloBio and Epiodyne. The other authors declare no competing interests.
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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 and 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 and 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 and 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 and 3: yeast display and OPS-RD binding data of PAC1R binders. Supplementary Tabes 4 and 5: pharmacological data of MRGPRX1 and NK1R binders. Supplementary Table 6: contact list between MRGPRX1 and adducts at a distance of <5 Å. Supplementary Tables 7 and 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.
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Muratspahić, E., Feldman, D., Kim, D.E. et al. De novo design of miniproteins targeting GPCRs. Nature 656, 1044–1053 (2026). https://doi.org/10.1038/s41586-026-10656-8
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DOI: https://doi.org/10.1038/s41586-026-10656-8
