{
  "abstract": "Glycine transporter 1 (GlyT1) is a clinically investigated target whose conformational plasticity enables ligands to engage distinct extracellular and intracellular binding sites. However, methods to directly quantify intracellular binding remain limited and largely rely on radioligand-based approaches. Here, we report a NanoBRET-based binding assay to quantify intracellular GlyT1 target engagement in living cells using a fluorescent tracer derived from the GlyT1 inhibitor Cmpd1. With a structure-based approach followed by pharmacological characterization we identified a suitable meta-substituted exit vector for linker and fluorophore attachment while maintaining GlyT1 binding. A series of fluorescent probes was synthesized and evaluated, leading to the development of GlyT1-probe which displayed high-affinity binding and enabled robust NanoBRET measurements. Unlike the functional uptake assay, the NanoBRET assay distinguished intracellular from extracellular GlyT1 inhibitors and resolved structure-activity relationships at the intracellular binding site. This assay provides a quantitative approach to interrogate conformation-selective GlyT1 target engagement in living cells, facilitating the characterization of GlyT1 ligands targeting the intracellular site.",
  "abstract_shown": true,
  "affiliations": [
    "University of Copenhagen"
  ],
  "article_type": "New results",
  "arxiv": null,
  "author_total": 9,
  "authors": [
    {
      "name": "M. H. Jensen"
    },
    {
      "name": "I. P. Avgeris"
    },
    {
      "name": "N. M. Kocaturk"
    },
    {
      "name": "A. N. L. Mikkelsen"
    },
    {
      "name": "B. Frolund"
    },
    {
      "name": "W. Farnaby"
    },
    {
      "name": "P. Wellendorph"
    },
    {
      "name": "J. S. H. Mortensen"
    },
    {
      "affs": [
        0
      ],
      "name": "F. Bavo"
    }
  ],
  "bibtex": "https://research.draconitic.com/paper/15fedkg07pbn0.bib",
  "cited_by": 0,
  "doi": "10.64898/2026.10.05.756331",
  "fulltext_url": "https://www.biorxiv.org/content/10.64898/2026.10.05.756331v1.full",
  "id": "15fedkg07pbn0",
  "issns": [],
  "landing_url": "https://www.biorxiv.org/content/10.64898/2026.10.05.756331v1",
  "language": null,
  "licence": "CC-BY-NC-ND",
  "licence_label": "CC BY-NC-ND",
  "licence_source": "article",
  "licence_url": "https://creativecommons.org/licenses/by-nc-nd/4.0/",
  "metadata_licence": null,
  "native_subjects": [
    [
      "biorxiv",
      "molecular biology"
    ],
    [
      "openalex",
      "1303|Amino Acid Enzymes and Metabolism"
    ]
  ],
  "openalex": "W7220709518",
  "pdf_url": "https://www.biorxiv.org/content/10.64898/2026.10.05.756331v1.full.pdf",
  "pmcid": null,
  "pmid": null,
  "published": "2026-10-05",
  "publisher": null,
  "ris": "https://research.draconitic.com/paper/15fedkg07pbn0.ris",
  "sources": [
    {
      "first_seen": "2026-10-30T12:26:16Z",
      "native_id": "10.64898/2026.10.05.756331",
      "source": "biorxiv",
      "url": "https://www.biorxiv.org/content/10.64898/2026.10.05.756331v1"
    }
  ],
  "subjects": [
    {
      "field": "biochemistry",
      "origin": "biorxiv:molecular biology",
      "subfield": "molecular-biology"
    }
  ],
  "title": "Development of a NanoBRET assay platform to detect intracellular target engagement in living cells for the Glycine Transporter 1 (SLC6A9)",
  "title_text": "Development of a NanoBRET assay platform to detect intracellular target engagement in living cells for the Glycine Transporter 1 (SLC6A9)",
  "type": "preprint",
  "updated_at": "2026-10-30T12:26:16Z",
  "url": "https://research.draconitic.com/paper/15fedkg07pbn0",
  "venue": "bioRxiv",
  "versions": []
}