An engineered nanobody inhibitor for molecular-to-circuit control of opioid receptor function

An engineered nanobody inhibitor for molecular-to-circuit control of opioid receptor function | bioRxiv Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results An engineered nanobody inhibitor for molecular-to-circuit control of opioid receptor function Zoé Valbret , Paul J. Lamothe-Molina , Sofia Papadogkonaki , Antoine Koehl , Jan Vincent V. Arafiles , Ziva Shapiro Tuchman , Nicole M. Fisher , Arthur Radoux-Mergault , Annika Canziani , Gaoyang Huang , Mark von Zastrow , Mickey Kosloff , Christian P. R. Hackenberger , Aashish Manglik , Tommaso Patriarchi , View ORCID Profile Miriam Stoeber doi: https://doi.org/10.64898/2026.01.12.698943 Zoé Valbret 1 Department of Cell Physiology and Metabolism, Faculty of Medicine, University of Geneva , Geneva, Switzerland Find this author on Google Scholar Find this author on PubMed Search for this author on this site Paul J. Lamothe-Molina 2 Institute of Pharmacology and Toxicology, University of Zurich , Zurich, Switzerland Find this author on Google Scholar Find this author on PubMed Search for this author on this site Sofia Papadogkonaki 1 Department of Cell Physiology and Metabolism, Faculty of Medicine, University of Geneva , Geneva, Switzerland Find this author on Google Scholar Find this author on PubMed Search for this author on this site Antoine Koehl 3 Department of Electrical Engineering and Computer Sciences, University of California , Berkeley, CA, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Jan Vincent V. Arafiles 4 Institut für Chemie, Humboldt-Universität zu Berlin , Berlin, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site Ziva Shapiro Tuchman 5 The Department of Human Biology, Faculty of Natural Sciences, University of Haifa , Haifa, Israel Find this author on Google Scholar Find this author on PubMed Search for this author on this site Nicole M. Fisher 6 Weill Institute for Neurosciences, Department of Psychiatry and Behavioral Sciences, University of California , San Francisco, San Francisco, CA, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Arthur Radoux-Mergault 1 Department of Cell Physiology and Metabolism, Faculty of Medicine, University of Geneva , Geneva, Switzerland Find this author on Google Scholar Find this author on PubMed Search for this author on this site Annika Canziani 2 Institute of Pharmacology and Toxicology, University of Zurich , Zurich, Switzerland Find this author on Google Scholar Find this author on PubMed Search for this author on this site Gaoyang Huang 2 Institute of Pharmacology and Toxicology, University of Zurich , Zurich, Switzerland Find this author on Google Scholar Find this author on PubMed Search for this author on this site Mark von Zastrow 6 Weill Institute for Neurosciences, Department of Psychiatry and Behavioral Sciences, University of California , San Francisco, San Francisco, CA, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Mickey Kosloff 5 The Department of Human Biology, Faculty of Natural Sciences, University of Haifa , Haifa, Israel Find this author on Google Scholar Find this author on PubMed Search for this author on this site Christian P. R. Hackenberger 4 Institut für Chemie, Humboldt-Universität zu Berlin , Berlin, Germany 7 Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP) , Berlin, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site Aashish Manglik 8 Department of Pharmaceutical Chemistry, University of California , San Francisco, San Francisco, CA, USA 9 Department of Anesthesia and Perioperative Care, University of California , San Francisco, San Francisco, CA, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: miriam.stoeber{at}unige.ch patriarchi{at}pharma.uzh.ch aashish.manglik{at}ucsf.edu Tommaso Patriarchi 2 Institute of Pharmacology and Toxicology, University of Zurich , Zurich, Switzerland 10 Neuroscience Center Zurich, University and ETH Zurich , Zurich, Switzerland Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: miriam.stoeber{at}unige.ch patriarchi{at}pharma.uzh.ch aashish.manglik{at}ucsf.edu Miriam Stoeber 1 Department of Cell Physiology and Metabolism, Faculty of Medicine, University of Geneva , Geneva, Switzerland Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Miriam Stoeber For correspondence: miriam.stoeber{at}unige.ch patriarchi{at}pharma.uzh.ch aashish.manglik{at}ucsf.edu Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract Opioid receptors (ORs) orchestrate pain relief, reward, and dependence, yet their signaling arises from diverse cell types and subcellular compartments that cannot be selectively interrogated with existing pharmacological or genetic approaches. Single-domain antibodies, or nanobodies (Nbs), can probe receptor states, but their potential as tools for controlling native receptor signaling at the system level has remained unexplored. Here, we engineer a suite of high-affinity intracellular Nbs that bind active ORs through structure-guided evolution and in silico design. Iterative optimization yields Nb64, a potent inhibitor that rapidly suppresses transducer engagement, receptor internalization, and downstream signaling, including endogenous pathways in neuronal cells. Organelle targeting highlights Nb64’s capacity to control OR activity with subcellular precision, while bio-reversible cell-penetrating peptide (CPP) conjugation enables non-genetic cytosolic delivery. Cell-type-specific expression of Nb64 in VTA interneurons attenuates fentanyl-evoked dopamine release and behavioral responses in mice, demonstrating targeted control of opioid actions in vivo. Nb64 provides a versatile strategy for dissecting OR biology and establishes a generalizable framework for precision inhibition of native GPCR signaling in vivo. Competing Interest Statement AM is a founder of Epiodyne, Stipple Bio, and BNM Oncology, and consults for Abalone, Alkermes, Kingfisher Therapeutics, and Septerna. MvZ consults for Deep Apple Therapeutics. The other authors declare no competing interests. Funder Information Declared National Institutes of Health , R61DA051531 Swiss National Science Foundation , PCEFP3_181282 , 320030E_224301 , 320030-236030 European Research Council , 891959 Israel Science Foundation, https://ror.org/04sazxf24 , 197/25 , 3960/25 Copyright The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license . Back to top Previous Next Posted January 14, 2026. 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Fisher , Arthur Radoux-Mergault , Annika Canziani , Gaoyang Huang , Mark von Zastrow , Mickey Kosloff , Christian P. R. Hackenberger , Aashish Manglik , Tommaso Patriarchi , Miriam Stoeber bioRxiv 2026.01.12.698943; doi: https://doi.org/10.64898/2026.01.12.698943 Share This Article: Copy Citation Tools An engineered nanobody inhibitor for molecular-to-circuit control of opioid receptor function Zoé Valbret , Paul J. Lamothe-Molina , Sofia Papadogkonaki , Antoine Koehl , Jan Vincent V. Arafiles , Ziva Shapiro Tuchman , Nicole M. Fisher , Arthur Radoux-Mergault , Annika Canziani , Gaoyang Huang , Mark von Zastrow , Mickey Kosloff , Christian P. R. Hackenberger , Aashish Manglik , Tommaso Patriarchi , Miriam Stoeber bioRxiv 2026.01.12.698943; doi: https://doi.org/10.64898/2026.01.12.698943 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Neuroscience Subject Areas All Articles Animal Behavior and Cognition (8164) Biochemistry (19027) Bioengineering (15171) Bioinformatics (45808) Biophysics (22958) Cancer Biology (20248) Cell Biology (27495) Clinical Trials (138) Developmental Biology (14233) Ecology (21529) Epidemiology (2067) Evolutionary Biology (25940) Genetics (16436) Genomics (23979) Immunology (19176) Microbiology (43488) Molecular Biology (18483) Neuroscience (95839) Paleontology (717) Pathology (3064) Pharmacology and Toxicology (5253) Physiology (8339) Plant Biology (16378) Scientific Communication and Education (2123) Synthetic Biology (4675) Systems Biology (10451) Zoology (2436)

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