Abstract
Difelikefalin is an FDA-approved κ-opioid receptor (KOR) peptide agonist used to treat chronic pruritus. However, as a balanced agonist that activates both G protein and β-arrestin pathways, difelikefalin remains associated with undesirable side effects linked to β-arrestin signaling. Here, we report the cryo-EM structure of the difelikefalin-KOR-Gi complex, identifying Y3207.43 as a key residue that is critical for signaling bias. Guided by this structural insight, we engineer beta01, a β-amino acid-substituted analog with potent G protein activation but minimal β-arrestin recruitment. In mouse models, beta01 retains robust antinociceptive and antipruritic efficacy while significantly reducing sedation and anxiety-like behaviors. Structural, molecular dynamics simulations and 2D 13C-Met NMR analyses further reveal beta01 stabilizes a unique KOR conformation with an expanded intracellular cavity that disfavors β-arrestin binding. This work establishes a rational structure-based framework for designing safer and more effective GPCR-targeted therapeutics.
Data availability
The cryo-EM density maps and atomic coordinates have been deposited in the Electron Microscopy Data Bank (EMDB) and Protein Data Bank (PDB) under accession numbers EMD-68217 and 22ES for the difelikefalin-KOR-Gi complex, and EMD-68208 and 22EM for the beta01-KOR-Gi complex. All the raw data from our molecular dynamic simulations have been uploaded to the public repository Zenodo (https://zenodo.org/records/17984759). The relevant source data from each figure or table in the main manuscript and in the Supplementary Information are provided as Source Data file. Source data are provided with this paper.
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Acknowledgements
The cryo-EM data were collected at the Advanced Center for Electron Microscopy, Shanghai Institute of Materia Medica (SIMM) and the Center for Integrative Imaging of University of Science and Technology of China (Hefei). Difelikefalin derivatives in this study were synthesized by Hefei KS-V Peptide Biological Technology Co. Ltd. This project was supported by the National Natural Science Foundation of China (22437005 to C.T., 22277114 to P.S.), the National Key Research & Development Project, Mimistry of Science and Technology the China (2022YFC3400500), the Strategic Priority Research Program of Chinese Academy of Sciences (XDB0960000 to C.T.), the “USTC Research Funds of the Double First-Class Initiative” (YD9100002021 to P.S.). The Foundation of National Facility for Translational Medicine (Shanghai)). The National Natural Science Foundation of China (32130022, 82495184, 82121005 to H.E.X., 32400998 to Y.W. 22477116 to F.Y.); the National Key R&D Program of China (2022YFA1302900 to W.Y; 2022YFC2703105 to H.E.X.); National Key R&D Program “Strategic Scientific and Technological Innovation Cooperation” Key Project (2022YFE0203600) released by the Ministry of Science and Technology; CAS Strategic Priority Research Program (XDB37030103 to H.E.X.); Shanghai Municipal Science and Technology Major Project (2019SHZDZX02 to H.E.X.); Shanghai Municipal Science and Technology Major Project (H.E.X.); Supported by the Strategic Priority Research Program of the Chinese Academy of Sciences; Grant No. XDB0830000 to H.E.X., the Special Research Assistant Project of Chinese Academy of Sciences (to Y.W.); the Young Elite Scientists Sponsorship Program by CAST (2023QNRC001 to Y.Z.); the Natural Science Foundation of Shanghai, China (23ZR1475300 to Y.Z.); the Sailing Program of Shanghai Venus Project (23YF1456700 to Y.Z.); the Youth Innovation Promotion Association of the Chinese Academy of Sciences (2023298 to Y.Z.). Beijing Life Science Academy Scientific Research Program (2023000CA0070, 2024100CA0060). National Facility for Translational Medicine (Shanghai) Open Research Program (TMSK-2024-117 to P.L.).
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H.Z., Y.W., and R.W. expressed, optimized and purified the receptor complex and prepared the cryo-EM grids. W.H. and K.W. performed cryo-EM data acquisition. Y.X. and Q.Y. determined the cryo-EM structures. Y.W., H.Z. and F.Y. analyzed the structures. P.S., H.Z., R.W. and Z.X. participated in Gαi-Gγ dissociation, arrestin recruitment assays. H.Z. and Q.Z. performed animal behavior experiments. Z.X. and H.Z prepared cell surface expression level analysis. G.W., X.H., Y.L. performed molecular dynamics simulations. P.L., Y.Z. and L.Z. performed peptide library design. J.L. and Y.Z. participated in chemical synthesis. Y.W., H.Z. and P.S. prepared the figures and participated manuscript writing; C.T., H.E.X., P.S. and Y.W wrote the manuscript with inputs from the authors. C.T., H.E.X., P.S. and Y.W. conceived and supervised the project.
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H.E.X is a founder of Cascade Pharmaceutics. C.T. is a founder of Hefei KS-V Peptide Biological Technology Co. Ltd.; All the other authors declare no other competing interests.
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Zhang, H., Wang, R., Shi, P. et al. Rational design of a Kappa opioid receptor peptide agonist with attenuated β-arrestin signaling. Nat Commun (2026). https://doi.org/10.1038/s41467-026-71455-3
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DOI: https://doi.org/10.1038/s41467-026-71455-3
