A reversible allosteric inhibitor of GlyT2 alleviates neuropathic pain without on-target side effects

  • bioRxiv. 2025 Nov 20:2025.04.21.649698. doi: 10.1101/2025.04.21.649698.
Ryan P Cantwell Chater  1  2 ,  Julian Peiser-Oliver  2 ,  Tanmay K Pati  3 ,  Ada S Quinn  4  5 ,  Irina Lotsaris  2 ,  Zachary J Frangos  6  7 ,  Kristen E Anderson  8  9 ,  Anna E Tischer  6 ,  Billy J Williams-Noonan  4  5 ,  Karin R Aubrey  8  9 ,  Megan L O'Mara  4  5 ,  Michael Michaelides  6 ,  Sarasa A Mohammadi  9  10 ,  Christopher L Cioffi  3 ,  Robert J Vandenberg  2 ,  Azadeh Shahsavar  1
Affiliations
  • 1. Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
  • 2. School of Medical Sciences, University of Sydney, Sydney, NSW, Australia.
  • 3. Department of Chemistry and Chemical Biology, Rensselaer Polytechnic Institute, Troy, New York, USA.
  • 4. Australian Institute of Bioengineering and Nanotechnology, The University of Queensland, St Lucia, Queensland, Australia.
  • 5. ARC Industry Transformation Training Centre for Cryo-electron Microscopy of Membrane Proteins (CCeMMP).
  • 6. Biobehavioral Imaging & Molecular Neuropsychopharmacology Unit, Neuroimaging Research Branch, National Institute on Drug Abuse, Intramural Research Program, Baltimore, MD, USA.
  • 7. Medication Development Program, National Institute on Drug Abuse, Intramural Research Program.
  • 8. Pain Management Research Institute, Kolling Institute, Royal North Shore Hospital NSLHD, St Leonards, New South Wales, Australia.
  • 9. Faculty of Medicine and Health, University of Sydney, Sydney, New South Wales, Australia.
  • 10. School of Pharmacy, University of Sydney, Sydney, NSW, Australia.
PMID: 41332641 DOI: 10.1101/2025.04.21.649698
Abstract

Chronic neuropathic pain, caused by nerve damage or disease, is increasing in prevalence, but current treatments are ineffective and over-reliant on opioids. The neuronal glycine transporter, GlyT2, regulates inhibitory glycinergic neurotransmission and represents a promising target for new analgesics. However, most GlyT2 inhibitors cause significant side effects, in part due to irreversible inhibition at analgesic doses. Here we develop a reversible inhibitor of GlyT2, RPI-GLYT2-82, and identify its binding site by determining cryo-EM structures of human GlyT2. We capture three fundamental conformational states of GlyT2 in the substrate-free state, and bound to either glycine, RPI-GLYT2-82 or the pseudo-irreversible inhibitor ORG25543. We demonstrate that RPI-GLYT2-82 dissociates from GlyT2 faster than ORG25543, providing analgesia in mouse neuropathic pain models without on-target side-effects or addiction liability. Our data provide a mechanistic understanding of allosteric inhibition of glycine transport, enabling structure-based design of non-opioid analgesics.

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