Development of a NanoBRET assay platform to detect intracellular target engagement in living cells for the Glycine Transporter 1 (SLC6A9)
- 1University of Copenhagen
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.
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BibTeX
@misc{jensen2026development,
title = {{Development of a NanoBRET assay platform to detect intracellular target engagement in living cells for the Glycine Transporter 1 (SLC6A9)}},
author = {M. H. Jensen and I. P. Avgeris and N. M. Kocaturk and A. N. L. Mikkelsen and B. Frolund and W. Farnaby and P. Wellendorph and J. S. H. Mortensen and F. Bavo},
howpublished = {bioRxiv},
year = {2026},
month = oct,
doi = {10.64898/2026.10.05.756331},
url = {https://www.biorxiv.org/content/10.64898/2026.10.05.756331v1},
note = {CC BY-NC-ND},
}
RIS
TY - UNPB
TI - Development of a NanoBRET assay platform to detect intracellular target engagement in living cells for the Glycine Transporter 1 (SLC6A9)
AU - M. H. Jensen
AU - I. P. Avgeris
AU - N. M. Kocaturk
AU - A. N. L. Mikkelsen
AU - B. Frolund
AU - W. Farnaby
AU - P. Wellendorph
AU - J. S. H. Mortensen
AU - F. Bavo
PY - 2026
DA - 2026/10/05
PB - bioRxiv
DO - 10.64898/2026.10.05.756331
UR - https://www.biorxiv.org/content/10.64898/2026.10.05.756331v1
AB - 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.
ER -