A Versatile Sub-Nanomolar Fluorescent Ligand Enables NanoBRET Binding Studies and Single-Molecule Microscopy at the Histamine H3Receptor

Niklas Rosier; Lukas Grätz; Hannes Schihada; Jan Möller; Ali Işbilir; Laura J. Humphrys; Martin Nagl; Ulla Seibel; Martin J. Lohse; Steffen Pockes

doi:10.1021/acs.jmedchem.1c01089

A Versatile Sub-Nanomolar Fluorescent Ligand Enables NanoBRET Binding Studies and Single-Molecule Microscopy at the Histamine H₃Receptor

Niklas Rosier, Lukas Grätz, Hannes Schihada, Jan Möller, Ali Işbilir, Laura J. Humphrys, Martin Nagl, Ulla Seibel, Martin J. Lohse, Steffen Pockes

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Abstract

The histamine H3 receptor (H3R) is considered an attractive drug target for various neurological diseases. We here report the synthesis of UR-NR266, a novel fluorescent H3R ligand. Broad pharmacological characterization revealed UR-NR266 as a sub-nanomolar compound at the H3R with an exceptional selectivity profile within the histamine receptor family. The presented neutral antagonist showed fast association to its target and complete dissociation in kinetic binding studies. Detailed characterization of standard H3R ligands in NanoBRET competition binding using UR-NR266 highlights its value as a versatile pharmacological tool to analyze future H3R ligands. The low nonspecific binding observed in all experiments could also be verified in TIRF and confocal microscopy. This fluorescent probe allows the highly specific analysis of native H3R in various assays ranging from optical high throughput technologies to biophysical analyses and single-molecule studies in its natural environment. An off-target screening at 14 receptors revealed UR-NR266 as a selective compound.

Original language	English
Pages (from-to)	11695-11708
Number of pages	14
Journal	Journal of Medicinal Chemistry
Volume	64
Issue number	15
DOIs	https://doi.org/10.1021/acs.jmedchem.1c01089
Publication status	Published - 12 Aug 2021
Externally published	Yes

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10.1021/acs.jmedchem.1c01089

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Rosier, N., Grätz, L., Schihada, H., Möller, J., Işbilir, A., Humphrys, L. J., Nagl, M., Seibel, U., Lohse, M. J., & Pockes, S. (2021). A Versatile Sub-Nanomolar Fluorescent Ligand Enables NanoBRET Binding Studies and Single-Molecule Microscopy at the Histamine H₃Receptor. Journal of Medicinal Chemistry, 64(15), 11695-11708. https://doi.org/10.1021/acs.jmedchem.1c01089

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title = "A Versatile Sub-Nanomolar Fluorescent Ligand Enables NanoBRET Binding Studies and Single-Molecule Microscopy at the Histamine H3Receptor",

abstract = "The histamine H3 receptor (H3R) is considered an attractive drug target for various neurological diseases. We here report the synthesis of UR-NR266, a novel fluorescent H3R ligand. Broad pharmacological characterization revealed UR-NR266 as a sub-nanomolar compound at the H3R with an exceptional selectivity profile within the histamine receptor family. The presented neutral antagonist showed fast association to its target and complete dissociation in kinetic binding studies. Detailed characterization of standard H3R ligands in NanoBRET competition binding using UR-NR266 highlights its value as a versatile pharmacological tool to analyze future H3R ligands. The low nonspecific binding observed in all experiments could also be verified in TIRF and confocal microscopy. This fluorescent probe allows the highly specific analysis of native H3R in various assays ranging from optical high throughput technologies to biophysical analyses and single-molecule studies in its natural environment. An off-target screening at 14 receptors revealed UR-NR266 as a selective compound.",

author = "Niklas Rosier and Lukas Gr{\"a}tz and Hannes Schihada and Jan M{\"o}ller and Ali I{\c s}bilir and Humphrys, {Laura J.} and Martin Nagl and Ulla Seibel and Lohse, {Martin J.} and Steffen Pockes",

note = "Funding Information: This work was supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation – 427840891) to H.S. Financial support by the Deutsche Forschungsgemeinschaft (DFG, Research Training Group GRK1910) is gratefully acknowledged (L.G., L.J.H.). Financial support by the graduate school “Receptor Dynamics” of the Elite Network of Bavaria (ENB) is gratefully acknowledged (N.R., H.S., J.M., A.I., M.N., S.P.). Financial support by the EU training network Oncornet and the SFB 1423 is gratefully acknowledged (A.I., M.J.L.). Publisher Copyright: {\textcopyright} 2021 American Chemical Society.",

year = "2021",

month = aug,

day = "12",

doi = "10.1021/acs.jmedchem.1c01089",

language = "English",

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Rosier, N, Grätz, L, Schihada, H, Möller, J, Işbilir, A, Humphrys, LJ, Nagl, M, Seibel, U, Lohse, MJ & Pockes, S 2021, 'A Versatile Sub-Nanomolar Fluorescent Ligand Enables NanoBRET Binding Studies and Single-Molecule Microscopy at the Histamine H₃Receptor', Journal of Medicinal Chemistry, vol. 64, no. 15, pp. 11695-11708. https://doi.org/10.1021/acs.jmedchem.1c01089

TY - JOUR

T1 - A Versatile Sub-Nanomolar Fluorescent Ligand Enables NanoBRET Binding Studies and Single-Molecule Microscopy at the Histamine H3Receptor

AU - Rosier, Niklas

AU - Grätz, Lukas

AU - Schihada, Hannes

AU - Möller, Jan

AU - Işbilir, Ali

AU - Humphrys, Laura J.

AU - Nagl, Martin

AU - Seibel, Ulla

AU - Lohse, Martin J.

AU - Pockes, Steffen

N1 - Funding Information: This work was supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation – 427840891) to H.S. Financial support by the Deutsche Forschungsgemeinschaft (DFG, Research Training Group GRK1910) is gratefully acknowledged (L.G., L.J.H.). Financial support by the graduate school “Receptor Dynamics” of the Elite Network of Bavaria (ENB) is gratefully acknowledged (N.R., H.S., J.M., A.I., M.N., S.P.). Financial support by the EU training network Oncornet and the SFB 1423 is gratefully acknowledged (A.I., M.J.L.). Publisher Copyright: © 2021 American Chemical Society.

PY - 2021/8/12

Y1 - 2021/8/12

N2 - The histamine H3 receptor (H3R) is considered an attractive drug target for various neurological diseases. We here report the synthesis of UR-NR266, a novel fluorescent H3R ligand. Broad pharmacological characterization revealed UR-NR266 as a sub-nanomolar compound at the H3R with an exceptional selectivity profile within the histamine receptor family. The presented neutral antagonist showed fast association to its target and complete dissociation in kinetic binding studies. Detailed characterization of standard H3R ligands in NanoBRET competition binding using UR-NR266 highlights its value as a versatile pharmacological tool to analyze future H3R ligands. The low nonspecific binding observed in all experiments could also be verified in TIRF and confocal microscopy. This fluorescent probe allows the highly specific analysis of native H3R in various assays ranging from optical high throughput technologies to biophysical analyses and single-molecule studies in its natural environment. An off-target screening at 14 receptors revealed UR-NR266 as a selective compound.

AB - The histamine H3 receptor (H3R) is considered an attractive drug target for various neurological diseases. We here report the synthesis of UR-NR266, a novel fluorescent H3R ligand. Broad pharmacological characterization revealed UR-NR266 as a sub-nanomolar compound at the H3R with an exceptional selectivity profile within the histamine receptor family. The presented neutral antagonist showed fast association to its target and complete dissociation in kinetic binding studies. Detailed characterization of standard H3R ligands in NanoBRET competition binding using UR-NR266 highlights its value as a versatile pharmacological tool to analyze future H3R ligands. The low nonspecific binding observed in all experiments could also be verified in TIRF and confocal microscopy. This fluorescent probe allows the highly specific analysis of native H3R in various assays ranging from optical high throughput technologies to biophysical analyses and single-molecule studies in its natural environment. An off-target screening at 14 receptors revealed UR-NR266 as a selective compound.

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U2 - 10.1021/acs.jmedchem.1c01089

DO - 10.1021/acs.jmedchem.1c01089

M3 - Article

C2 - 34309390

AN - SCOPUS:85112693456

SN - 0022-2623

VL - 64

SP - 11695

EP - 11708

JO - Journal of Medicinal Chemistry

JF - Journal of Medicinal Chemistry

IS - 15

ER -

A Versatile Sub-Nanomolar Fluorescent Ligand Enables NanoBRET Binding Studies and Single-Molecule Microscopy at the Histamine H3Receptor

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A Versatile Sub-Nanomolar Fluorescent Ligand Enables NanoBRET Binding Studies and Single-Molecule Microscopy at the Histamine H₃Receptor